Post on 24-Jul-2020
Research ArticleAssessment of Functional EST-SSR Markers(Sugarcane) in Cross-Species Transferability Genetic Diversityamong Poaceae Plants and Bulk Segregation Analysis
Shamshad Ul Haq123 Pradeep Kumar14 R K Singh1 Kumar Sambhav Verma5
Ritika Bhatt23 Meenakshi Sharma3 Sumita Kachhwaha3 and S L Kothari235
1Biotechnology Division UP Council of Sugarcane Research Shahjahanpur 242001 India2Interdisciplinary Programme of Life Science for Advance Research and Education University of Rajasthan Jaipur 302004 India3Department of Botany University of Rajasthan Jaipur 302015 India4School of Biotechnology Yeungnam University Gyeongsan 712-749 Republic of Korea5Amity Institute of Biotechnology Amity University Rajasthan Jaipur 302006 India
Correspondence should be addressed to Shamshad Ul Haq shamshadbiotechgmailcom
Received 26 November 2015 Revised 13 April 2016 Accepted 26 April 2016
Academic Editor Norman A Doggett
Copyright copy 2016 Shamshad Ul Haq et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Expressed sequence tags (ESTs) are important resource for gene discovery gene expression and its regulation molecular markerdevelopment and comparative genomics We procured 10000 ESTs and analyzed 267 EST-SSRs markers through computationalapproach The average density was one SSR1045 kb or 64 frequency wherein trinucleotide repeats (6674) were the mostabundant followed by di- (2610) tetra- (467) penta- (15) and hexanucleotide (12) repeats Functional annotations weredone and after-effect newly developed 63 EST-SSRs were used for cross transferability genetic diversity and bulk segregationanalysis (BSA) Out of 63 EST-SSRs 42 markers were identified owing to their expansion genetics across 20 different plants whichamplified 519 alleles at 180 loci with an average of 288 alleleslocus and the polymorphic information content (PIC) ranged from051 to 093 with an average of 083The cross transferability ranged from 25 for wheat to 9722 for Schlerostachya with an averageof 5586 and genetic relationships were established based on diversification among themMoreover 10 EST-SSRs were recognizedas important markers between bulks of pooled DNA of sugarcane cultivars through BSA This study highlights the employabilityof the markers in transferability genetic diversity in grass species and distinguished sugarcane bulks
1 Introduction
Sugarcane is a bioenergy crop belonging to the genus Sac-charum L of the tribe Andropogoneae (family Poaceae)This tribe comprises grass species which have high eco-nomic value The noble sugarcane varieties are developedfrom interspecific hybridization of Saccharum officinarum L(2119899 = 80) which has high sugar content with less diseasetolerance and Saccharum spontaneum (2119899 = 40 to 120) whichprovides stress disease tolerance and high fiber content forbiomassThe taxonomy and genetic constitution of sugarcaneare complicated due to complex interspecific aneupolyploidgenome which makes chromosome numbers range from 100
to 130 [1] Moreover six Saccharum spp (S spontaneum Sofficinarum S robustum S edule S barberi and S sinense)and four Saccharum related genera (Erianthus MiscanthusSclerostachya and Narenga) have purportedly undergoneinterbreeding forming the ldquoSaccharum complexrdquo [2 3] Theinterbreeding has made their genome more complex andadded tomultigenic andor multiallelic nature for most agro-nomic traits that made sugarcane breeding a more difficulttask [4]
A vast array of genomic tools has been developed whichhas opened new ways to define the genetic architecture ofsugarcane and helped to explore its functional system [15] Among the molecular markers microsatellites are most
Hindawi Publishing CorporationGenetics Research InternationalVolume 2016 Article ID 7052323 16 pageshttpdxdoiorg10115520167052323
2 Genetics Research International
favored for a variety of genetic applications due to theirmultiallelic nature high reproducibility cross transferabilitycodominant inheritance abundance and extensive genomecoverage [6ndash8] Microsatellites or simple sequences repeats(SSRs) are monotonous repetitions of very short (one tosix) nucleotide motifs which occur as interspersed repet-itive elements in all eukaryotic and prokaryotic genomesHowever transcribed regions of the genome also containenormous range of microsatellites that correspond to genicmicrosatellites or EST-SSRs Therefore expressed sequencetags (ESTs) are the short transcribed portions and involvedin the variety of metabolic functions The presence of themicrosatellites in genes as well as ESTs unveils the biologicalsignificance of SSR distribution expansion and contractionon the function of the genes themselves [9]
Presently huge amounts of expressed sequence tagshave been deposited in public database (NCBI) In silicoapproaches to retrieve EST sequences from NCBI and func-tional annotations provide more constructive EST-SSRs orgene-based SSR (genic SSRs) marker development besidesown EST libraries developmentThis method of the EST-SSRmarkers development provides the easiest way to reduce costtime and labours along with more meaningful marker iden-tifications [10] The presence of microsatellites in the genicregion is found to be more conserved due to which they pos-sess high reproducibility and high interspecificintraspecifictransferability Hence EST-SSR could be used for polymor-phism genetic diversity cross transferability and compara-tive mapping in different plant species Accordingly severalgenetic studies were done on sugarcane using microsatellitemarkers to decipher polymorphism cross transferabilitygenetic diversity informative marker detection through bulksegregation analysis (BSA) and comparative genomics [811ndash13] The objective of the present study was to retrieveEST sequences for more informative EST-SSR developmentand their genetic assessment within and across the taxathrough cross transferability genetic relationships and bulksegregation analysis
2 Materials and Methods
21 EST Sequences Retrieving ESTs Assembling andMicrosatellites Identification Total 10000 EST sequences ofthe Saccharum spp were downloaded in Fasta format fromNational Centre for Biotechnology Information (NCBI) formicrosatellites deciphering Further ESTs assembling wascarried out using CAP3 programme (httpmobylepasteurfrcgi-binportalpyformscap3) for minimization of se-quences redundancyMicrosatellite identificationwas carriedout using MISA software (httppgrcipk-gaterslebendemisa) and the criteria for SSR detection were 6 4 3 3and 3 repeat units for di- tri- tetra- penta- and hexanu-cleotides respectively SSR primer pairs (forward andreverse) were designed for the selected EST sequences havingmicrosatellites using online web tool batch primer 3 pipeline[14]
22 EST-SSR Sequences Annotation Assessment of ESTsequences having SSR was done through blastnblastx
analysis for homology search and against nonredundant (nr)protein at the NCBI Furthermore functional annotationpipeline was also run at online tool for gene ontology (GO)which was intended for different GO functional classeslike biological process cellular component and molecularfunction [15]
23 PCR Amplification and Electrophoresis PCR reactionswere carried out in a total of 10 120583L volume containing 25 ngtemplate DNA 10 120583L (10 pmol120583L) of each forward andreverse primer 100mM of dNTPs 05U of Taq DNA poly-merase and 10 120583L of 10x PCR buffer with 25mM of MgCl
2
Amplification was performed in a thermal cycler (Bio-Rad)in the following conditions initial denaturation at 94∘C for5min followed by 30 amplification cycles of denaturation for1min at 94∘C followed by annealing temperature (119879
119886) for
1min and then extension for 2min at 72∘C final extension at72∘C for 7min was allowed The PCR conditions particularlythe annealing temperatures (varying from 52∘C to 58∘C) foreach primer were standardized and amplified products werestored at 4∘C The PCR products were analyzed on a 7native PAGE in vertical gel electrophoresis unit (BangaloreGenei) using TBE buffer The sizes of amplified fragmentswere estimated using 50 bp DNA ladder (Fermentas) Gelswere documented using ethidium bromide (EtBr) staineddye
24 Evaluation of SaccharumEST-SSR across the Taxa throughCross Transferability The cross transferability of Saccharumderived EST-SSR markers was evaluated among the 20accessions comprising seven cereals (wheat maize barleyrice pearl millet oat and Sorghum) four Saccharum relatedgenera (Erianthus Miscanthus Narenga and Sclerostachya)three Saccharum species (51NG56 (S robustum) N58 (Sspontaneum) and two clones of S officinarum (BandjermasinHitam and Gunjera)) and five Saccharum commercial culti-vars (CoS 88230 CoS 92423 UP 9530 CoS 8436 and CoS91230) All genotypes were collected from the SugarcaneResearch Institute Farm UPCSR Shahjahanpur India Fur-thermore genomic DNA from young juvenile disease-freeimmature leaves was isolated for each genotype using CTAB(cetyl trimethylammonium bromide) method [16] IsolatedDNA samples were treated with RNAase for 1 h at 37∘C andpurified by phenol extraction (25 phenol 24 chloroform 1isoamyl alcohol vvv) followed by ethanol precipitation [17]and stored at minus80∘C DNAwas quantified on 08 agarose geland the working concentration of 25 ng120583L was obtained bymaking final adjustment in 10mM TE buffer
25 Genetic Diversity Analysis The assessment of EST-SSRsin genetic diversity analysis was done among 20 plantsbelonging to distinct groups comprising cereals Saccharumrelated genera Saccharum species and Saccharum cultivarsThe allelic data of 63 EST-SSR primers were used to ascertainthe genetic relationships between 20 genotypes by clusteringanalysis Amplified bands were scored as binary data in theform of present (1) or absent (0) Dendrogram was con-structed by neighbour-joining and Jaccardrsquos algorithm using
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FreeTree and TreeView software [18 19] The polymorphicinformation content (PIC) values were calculated for eachprimer by using the online resource of PIC Calculator (httpwwwlivacuksimkempsjpichtml)
26 Informative Assessment of Functional EST-SSR Markersbetween Bulks Plantmaterials were used as F2mapping pop-ulation comprising 209 genotypes of the sugarcane cultivarswhichwere developed from cross betweenCoS 91230 (ParentCoS 775 times Co 1148) with CoS 8436 (Parent MS 6847 timesCo 1148) from September to March (2010-2011) Groupingof genotypes was done according to their stem diameter(contrasting high and low stem diameter genotypes) intotwo sets DNA extractions were carried out from both setsand equal quantities of genomic DNA from 10 extreme highstem diameter and 10 extreme low stem diameter genotypeswere pooled into two bulks PCR amplification was donein both bulks with newly developed EST-SSR primers forinformativemarkers identifications through bulk segregationanalysis (BSA) [20]
3 Results and Discussion
31 Mining of Microsatellites in EST Sequences and SSRsCharacterization Total 10000 EST sequences related toSaccharum spp were examined from NCBI for the simplesequence repeat (SSR) identification and characterizationusing computational approach Prior to the marker decipher-ing sequence assembly was performed and 6201 (4201 kb)nonredundant sequences were detected comprising 1752contigs and 4449 singlets wherein 406 SSRs were identifiedwith 360 perfect SSRs and 37 sequences containing morethan 1 SSR and 30 SSRs in compound formation There-fore computational and experimental approach to ascertainmicrosatellites in EST libraries from public database (NCBI)turned to be very cost effective and reduces time and labourbesides expense of own libraries development EST-SSRs area more preferable DNA marker in the variety of geneticanalysis and found to be more conserved as present in thetranscribed region of the genome These were found to bemore transferable across the taxonomic boundaries and couldbe evaluated as most informative markers for variety ofgenomics applications [10 21] These are more adapted inplants comparative genetic analysis for gene identificationgene mapping marker-assisted-selection transferability andgenetic diversity [7 22ndash24] Also a variety of studies havebeen reported on sugarcane using EST-SSR markers fordesired genetic analysis [8 13 25 26]
The frequency of SSR in EST sequences was 64 includ-ing all the repeats except mononucleotide repeats This resultis comparatively higher compared to previous studies onsugarcane [8 27ndash29] Contrary to this Singh et al [13]reported higher frequency (93) in sugarcane Kumpatlaand Mukhopadhyay [30] also observed high range (265 to1062) of SSR frequency in different plant species In generalabout 5 of ESTs contained SSR which has been reportedin many plant species [31] These variations in microsatellitefrequency could be attributed to the ldquosearch criteriardquo usedtype of SSRmotif size of sequence data and the mining tools
used [24 32] In otherwords the density of themicrosatelliteswas one SSR per 1045 kb which is closely comparable toearlier studies in sugarcane with densities 1 SSR109 kb [8]and 19 kb SSR [13]
Analysis revealed that trinucleotide repeats (6674)were found to be more frequent followed by di- (2610)tetra- (467) penta- (15) and hexanucleotide (12)repeats Our observation of high frequency of trinu-cleotide repeats is in agreement with previous reports onsugarcane [8 13 27ndash29 33] Several other studies havealso represented high frequency of trinucleotide repeatsin different plant species [24 31 34ndash36] A total of 33different types of motifs were identified of which fourbelonged to dinucleotide eight belonged to trinucleotidestwelve belonged to tetranucleotide five belonged to pen-tanucleotide and two belonged to hexanucleotide repeats(Figure 1) We observed that motifs AGCT and ATATwere more frequent in dinucleotide repeat followed bymotifs CCGCGGAGCCTGAGGCCT andACGCGT intrinucleotide repeat motif AAAGCTTT in tetranucleotiderepeats motif ACAGGCCTGT in pentanucleotide repeatsand AACACCGGTGTT in hexanucleotide repeats Thepresence of motif CCGCGG was also observed in sugarcaneby different authors [13 27] Kantety et al [37] also reportedCCGCGG motif as most abundant in wheat and SorghumSimilarly both Lawson and Zhang [38] and Da Maia etal [39] also observed abundance of motif CCGCGG indifferent member of the grass family Victoria et al [35] alsodecoded motif CCGCGG in the lower plants (C reinhardtiiand P patens) Thus this predominance of CCGCGG motiffrequency has been related to a high GC-content [5] Somemotifs which are responsible for making unusual DNAfolding structure (hairpin formed bipartite triplex formedand simple loop folding) also have effect on gene expressionsand regulationsmechanism namely CCTAGG CCGGGCGGATTC and GAATTC motifs [40 41] Moreover thepresence of trinucleotide repeats in the coding region formeda distinct group and encoded amino acid tracts within thepeptide [42] We also observed predictable twenty differenttypes of amino acids including stop codon Alanine arginineglycine proline and serine were most frequent (Figure 2)This is in agreement with previous studies that reported ondifferent plant species [11 35 43]
32 Expressed Sequence Tags Annotation and PrimersDevelopment All EST sequences having SSRswere examinedby functional annotation (blastn blastx and gene ontology)After-effect sixty-three ESTs having SSRs were successfullyidentified on the basis of their involvement in the variousmetabolic processes (Figure 3) After-effect sixty-threeEST-SSRs primer pairs were designed for polymorphicnature cross transferability bulk segregation analysis andgenetic diversity in the test plants (Table 1) These selectedEST-SSRs comprised all types of repeat motifs (excludingmononucleotide repeat) and among trinucleotide repeatsthey were highly frequent with GCTCGA TCCAGGand GGTCCA repeat motifs Similarly Sharma et al [44]also used functional annotation pipelines for the moreprominent molecular markers development related to gene
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Table 1 Details of selected 63 EST-SSR primer pairs used for cross transferability genetic diversity and bulks segregation analysis
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS28 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
081 939E minus 42 Protein transport proteinSec61 beta
SYMS28 R GTGTAGAACTGGAGCATTGAG
SYMS29 F GGGCAAGCAAGAAACCAC 52 (TCC)4
091 162E minus 24 Protein translation factorSUI1
SYMS29 R GAAGAGGTCAACCAAGAACTCSYMS30 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4086 100E minus 21 Preprotein translocase Sec
SYMS30 R GTGTAGAACTGGAGCATTGAG
SYMS31 F GAAGCTCCCAAGCTGCTA 53 (AGCT)3
076 200E minus 12 Predicted uncharacterizedprotein
SYMS31 R CCTACAGGAAAGATTTTAGGG
SYMS32 F GTCTCTTCTCCAGTTCTCCTT 55 (TGCG)4
084 246E minus 63Predictedactin-depolymerizingfactor
SYMS32 R GCTCAACAAATGTCTCCCTA
SYMS33 F TGCACTAACATGGTTGATGT 54 (GAAG)3
086 282E minus 90 Hypothetical proteinSORBIDRAFT 03g046450
SYMS33 R GGTGATTGTAAGGGTCATCTT
SYMS34 F GTTAATGGTGGTTCCGTTC 53 (GGC)6
088 4E minus 20 Predicted uncharacterizedprotein LOC101783547
SYMS34 R ATTATCAGCGCAGAGACATCSYMS35 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)
4075 100E minus 21 Preprotein translocase
SYMS35 R GTGTAGAACTGGAGCATTGAG
SYMS36 F GGACTGTACAAGGACGACAG 53 (GCT)4
070 114E minus 41 Protein transport proteinSec61 beta subunit
SYMS36 R TCTGCTTTCTTGGATATGGTA
SYMS37 F AAGAAGGATGCAAAGAAGAAG 54 (GAT)4
090 308E minus 81 Hypothetical proteinSORBIDRAFT 03g046450
SYMS37 R AGGCTTAGTAACAGCAGGTTTSYMS38 F AAGAAGGATGCAAAGAAGAAG 56 (AGA)
4086 900E minus 37 Hypothetical protein
SYMS38 R AGGCTTAGTAACAGCAGGTTTSYMS39 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 114E minus 41 Protein transport protein
SYMS39 R TCTGCTTTCTTGGATATGGTASYMS40 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 125E minus 40 Preprotein translocase
SYMS40 R TCTGCTTTCTTGGATATGGTA
SYMS41 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 962E minus 42 Protein transport proteinSec61 beta subunit
SYMS41 R TCTGCTTTCTTGGATATGGTASYMS42 F CCAAAGAGATCTTGCAGACTA mdash (ATG)
4mdash 178E minus 53 Jasmonate-induced protein
SYMS42 R CCCAACACAACAACCAATSYMS43 F CCACACAAGCAAGAAATAAAC mdash (GGT)
4mdash 857E minus 74 Dirigent-like protein
SYMS43 R TCGAACACTATGGTAAAGGTG
SYMS44 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41 Homeodomain-liketranscription factor
SYMS44 R TCTGCTTTCTTGGATATGGTASYMS45 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4069 986E minus 42 Protein transport protein
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Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS45 R GACTCTGCTTTCTTGGATATG
SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4
090 182E minus 44 Hypothetical proteinSORBIDRAFT 09g006220
SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)
3077 100E minus 21 Preprotein translocase Sec
SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)
6090 400E minus 20 TPA hypothetical protein
SYMS48 R CGTAAATATGAACGAGGTCAG
SYMS49 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein
SYMS49 R TCTGCTTTCTTGGATATGGTA
SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10
mdash 679E minus 13 TPA seed maturationprotein
SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)
4mdash 122E minus 41 Hypothetical protein
SYMS51 R ATTGTCACTTGCTATCCATTT
SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4
mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit
SYMS52 R GTAGATACCGAGCACACCAG
SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3
087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein
SYMS53 R GGATAGACTGAAATCTGCTCA
SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5
mdash 413E minus 08 Protein transport proteinSEC31
SYMS54 R GGAGGTGGAACTTCCTGA
SYMS55 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS55 R TCTGCTTTCTTGGATATGGTA
SYMS56 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)
6mdash 784E minus 27 Caltractin
SYMS57 R ATCAAAGAGATCAAAGGCTTC
SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6
074 597E minus 66Zinc finger A20 and AN1domains-containingprotein
SYMS58 R TAGGCTGCACAACAATAGTCT
SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6
080 402E minus 65 Predicted reticulon-likeprotein B1
SYMS59 R CAAGTACTCCAGCAGAGATGT
SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5
mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein
SYMS60 R TGTCACTAACACGAATCACAA
SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5
079 414E minus 57 Actin-depolymerizingfactor 3
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
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Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
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[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
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2 Genetics Research International
favored for a variety of genetic applications due to theirmultiallelic nature high reproducibility cross transferabilitycodominant inheritance abundance and extensive genomecoverage [6ndash8] Microsatellites or simple sequences repeats(SSRs) are monotonous repetitions of very short (one tosix) nucleotide motifs which occur as interspersed repet-itive elements in all eukaryotic and prokaryotic genomesHowever transcribed regions of the genome also containenormous range of microsatellites that correspond to genicmicrosatellites or EST-SSRs Therefore expressed sequencetags (ESTs) are the short transcribed portions and involvedin the variety of metabolic functions The presence of themicrosatellites in genes as well as ESTs unveils the biologicalsignificance of SSR distribution expansion and contractionon the function of the genes themselves [9]
Presently huge amounts of expressed sequence tagshave been deposited in public database (NCBI) In silicoapproaches to retrieve EST sequences from NCBI and func-tional annotations provide more constructive EST-SSRs orgene-based SSR (genic SSRs) marker development besidesown EST libraries developmentThis method of the EST-SSRmarkers development provides the easiest way to reduce costtime and labours along with more meaningful marker iden-tifications [10] The presence of microsatellites in the genicregion is found to be more conserved due to which they pos-sess high reproducibility and high interspecificintraspecifictransferability Hence EST-SSR could be used for polymor-phism genetic diversity cross transferability and compara-tive mapping in different plant species Accordingly severalgenetic studies were done on sugarcane using microsatellitemarkers to decipher polymorphism cross transferabilitygenetic diversity informative marker detection through bulksegregation analysis (BSA) and comparative genomics [811ndash13] The objective of the present study was to retrieveEST sequences for more informative EST-SSR developmentand their genetic assessment within and across the taxathrough cross transferability genetic relationships and bulksegregation analysis
2 Materials and Methods
21 EST Sequences Retrieving ESTs Assembling andMicrosatellites Identification Total 10000 EST sequences ofthe Saccharum spp were downloaded in Fasta format fromNational Centre for Biotechnology Information (NCBI) formicrosatellites deciphering Further ESTs assembling wascarried out using CAP3 programme (httpmobylepasteurfrcgi-binportalpyformscap3) for minimization of se-quences redundancyMicrosatellite identificationwas carriedout using MISA software (httppgrcipk-gaterslebendemisa) and the criteria for SSR detection were 6 4 3 3and 3 repeat units for di- tri- tetra- penta- and hexanu-cleotides respectively SSR primer pairs (forward andreverse) were designed for the selected EST sequences havingmicrosatellites using online web tool batch primer 3 pipeline[14]
22 EST-SSR Sequences Annotation Assessment of ESTsequences having SSR was done through blastnblastx
analysis for homology search and against nonredundant (nr)protein at the NCBI Furthermore functional annotationpipeline was also run at online tool for gene ontology (GO)which was intended for different GO functional classeslike biological process cellular component and molecularfunction [15]
23 PCR Amplification and Electrophoresis PCR reactionswere carried out in a total of 10 120583L volume containing 25 ngtemplate DNA 10 120583L (10 pmol120583L) of each forward andreverse primer 100mM of dNTPs 05U of Taq DNA poly-merase and 10 120583L of 10x PCR buffer with 25mM of MgCl
2
Amplification was performed in a thermal cycler (Bio-Rad)in the following conditions initial denaturation at 94∘C for5min followed by 30 amplification cycles of denaturation for1min at 94∘C followed by annealing temperature (119879
119886) for
1min and then extension for 2min at 72∘C final extension at72∘C for 7min was allowed The PCR conditions particularlythe annealing temperatures (varying from 52∘C to 58∘C) foreach primer were standardized and amplified products werestored at 4∘C The PCR products were analyzed on a 7native PAGE in vertical gel electrophoresis unit (BangaloreGenei) using TBE buffer The sizes of amplified fragmentswere estimated using 50 bp DNA ladder (Fermentas) Gelswere documented using ethidium bromide (EtBr) staineddye
24 Evaluation of SaccharumEST-SSR across the Taxa throughCross Transferability The cross transferability of Saccharumderived EST-SSR markers was evaluated among the 20accessions comprising seven cereals (wheat maize barleyrice pearl millet oat and Sorghum) four Saccharum relatedgenera (Erianthus Miscanthus Narenga and Sclerostachya)three Saccharum species (51NG56 (S robustum) N58 (Sspontaneum) and two clones of S officinarum (BandjermasinHitam and Gunjera)) and five Saccharum commercial culti-vars (CoS 88230 CoS 92423 UP 9530 CoS 8436 and CoS91230) All genotypes were collected from the SugarcaneResearch Institute Farm UPCSR Shahjahanpur India Fur-thermore genomic DNA from young juvenile disease-freeimmature leaves was isolated for each genotype using CTAB(cetyl trimethylammonium bromide) method [16] IsolatedDNA samples were treated with RNAase for 1 h at 37∘C andpurified by phenol extraction (25 phenol 24 chloroform 1isoamyl alcohol vvv) followed by ethanol precipitation [17]and stored at minus80∘C DNAwas quantified on 08 agarose geland the working concentration of 25 ng120583L was obtained bymaking final adjustment in 10mM TE buffer
25 Genetic Diversity Analysis The assessment of EST-SSRsin genetic diversity analysis was done among 20 plantsbelonging to distinct groups comprising cereals Saccharumrelated genera Saccharum species and Saccharum cultivarsThe allelic data of 63 EST-SSR primers were used to ascertainthe genetic relationships between 20 genotypes by clusteringanalysis Amplified bands were scored as binary data in theform of present (1) or absent (0) Dendrogram was con-structed by neighbour-joining and Jaccardrsquos algorithm using
Genetics Research International 3
FreeTree and TreeView software [18 19] The polymorphicinformation content (PIC) values were calculated for eachprimer by using the online resource of PIC Calculator (httpwwwlivacuksimkempsjpichtml)
26 Informative Assessment of Functional EST-SSR Markersbetween Bulks Plantmaterials were used as F2mapping pop-ulation comprising 209 genotypes of the sugarcane cultivarswhichwere developed from cross betweenCoS 91230 (ParentCoS 775 times Co 1148) with CoS 8436 (Parent MS 6847 timesCo 1148) from September to March (2010-2011) Groupingof genotypes was done according to their stem diameter(contrasting high and low stem diameter genotypes) intotwo sets DNA extractions were carried out from both setsand equal quantities of genomic DNA from 10 extreme highstem diameter and 10 extreme low stem diameter genotypeswere pooled into two bulks PCR amplification was donein both bulks with newly developed EST-SSR primers forinformativemarkers identifications through bulk segregationanalysis (BSA) [20]
3 Results and Discussion
31 Mining of Microsatellites in EST Sequences and SSRsCharacterization Total 10000 EST sequences related toSaccharum spp were examined from NCBI for the simplesequence repeat (SSR) identification and characterizationusing computational approach Prior to the marker decipher-ing sequence assembly was performed and 6201 (4201 kb)nonredundant sequences were detected comprising 1752contigs and 4449 singlets wherein 406 SSRs were identifiedwith 360 perfect SSRs and 37 sequences containing morethan 1 SSR and 30 SSRs in compound formation There-fore computational and experimental approach to ascertainmicrosatellites in EST libraries from public database (NCBI)turned to be very cost effective and reduces time and labourbesides expense of own libraries development EST-SSRs area more preferable DNA marker in the variety of geneticanalysis and found to be more conserved as present in thetranscribed region of the genome These were found to bemore transferable across the taxonomic boundaries and couldbe evaluated as most informative markers for variety ofgenomics applications [10 21] These are more adapted inplants comparative genetic analysis for gene identificationgene mapping marker-assisted-selection transferability andgenetic diversity [7 22ndash24] Also a variety of studies havebeen reported on sugarcane using EST-SSR markers fordesired genetic analysis [8 13 25 26]
The frequency of SSR in EST sequences was 64 includ-ing all the repeats except mononucleotide repeats This resultis comparatively higher compared to previous studies onsugarcane [8 27ndash29] Contrary to this Singh et al [13]reported higher frequency (93) in sugarcane Kumpatlaand Mukhopadhyay [30] also observed high range (265 to1062) of SSR frequency in different plant species In generalabout 5 of ESTs contained SSR which has been reportedin many plant species [31] These variations in microsatellitefrequency could be attributed to the ldquosearch criteriardquo usedtype of SSRmotif size of sequence data and the mining tools
used [24 32] In otherwords the density of themicrosatelliteswas one SSR per 1045 kb which is closely comparable toearlier studies in sugarcane with densities 1 SSR109 kb [8]and 19 kb SSR [13]
Analysis revealed that trinucleotide repeats (6674)were found to be more frequent followed by di- (2610)tetra- (467) penta- (15) and hexanucleotide (12)repeats Our observation of high frequency of trinu-cleotide repeats is in agreement with previous reports onsugarcane [8 13 27ndash29 33] Several other studies havealso represented high frequency of trinucleotide repeatsin different plant species [24 31 34ndash36] A total of 33different types of motifs were identified of which fourbelonged to dinucleotide eight belonged to trinucleotidestwelve belonged to tetranucleotide five belonged to pen-tanucleotide and two belonged to hexanucleotide repeats(Figure 1) We observed that motifs AGCT and ATATwere more frequent in dinucleotide repeat followed bymotifs CCGCGGAGCCTGAGGCCT andACGCGT intrinucleotide repeat motif AAAGCTTT in tetranucleotiderepeats motif ACAGGCCTGT in pentanucleotide repeatsand AACACCGGTGTT in hexanucleotide repeats Thepresence of motif CCGCGG was also observed in sugarcaneby different authors [13 27] Kantety et al [37] also reportedCCGCGG motif as most abundant in wheat and SorghumSimilarly both Lawson and Zhang [38] and Da Maia etal [39] also observed abundance of motif CCGCGG indifferent member of the grass family Victoria et al [35] alsodecoded motif CCGCGG in the lower plants (C reinhardtiiand P patens) Thus this predominance of CCGCGG motiffrequency has been related to a high GC-content [5] Somemotifs which are responsible for making unusual DNAfolding structure (hairpin formed bipartite triplex formedand simple loop folding) also have effect on gene expressionsand regulationsmechanism namely CCTAGG CCGGGCGGATTC and GAATTC motifs [40 41] Moreover thepresence of trinucleotide repeats in the coding region formeda distinct group and encoded amino acid tracts within thepeptide [42] We also observed predictable twenty differenttypes of amino acids including stop codon Alanine arginineglycine proline and serine were most frequent (Figure 2)This is in agreement with previous studies that reported ondifferent plant species [11 35 43]
32 Expressed Sequence Tags Annotation and PrimersDevelopment All EST sequences having SSRswere examinedby functional annotation (blastn blastx and gene ontology)After-effect sixty-three ESTs having SSRs were successfullyidentified on the basis of their involvement in the variousmetabolic processes (Figure 3) After-effect sixty-threeEST-SSRs primer pairs were designed for polymorphicnature cross transferability bulk segregation analysis andgenetic diversity in the test plants (Table 1) These selectedEST-SSRs comprised all types of repeat motifs (excludingmononucleotide repeat) and among trinucleotide repeatsthey were highly frequent with GCTCGA TCCAGGand GGTCCA repeat motifs Similarly Sharma et al [44]also used functional annotation pipelines for the moreprominent molecular markers development related to gene
4 Genetics Research International
Table 1 Details of selected 63 EST-SSR primer pairs used for cross transferability genetic diversity and bulks segregation analysis
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS28 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
081 939E minus 42 Protein transport proteinSec61 beta
SYMS28 R GTGTAGAACTGGAGCATTGAG
SYMS29 F GGGCAAGCAAGAAACCAC 52 (TCC)4
091 162E minus 24 Protein translation factorSUI1
SYMS29 R GAAGAGGTCAACCAAGAACTCSYMS30 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4086 100E minus 21 Preprotein translocase Sec
SYMS30 R GTGTAGAACTGGAGCATTGAG
SYMS31 F GAAGCTCCCAAGCTGCTA 53 (AGCT)3
076 200E minus 12 Predicted uncharacterizedprotein
SYMS31 R CCTACAGGAAAGATTTTAGGG
SYMS32 F GTCTCTTCTCCAGTTCTCCTT 55 (TGCG)4
084 246E minus 63Predictedactin-depolymerizingfactor
SYMS32 R GCTCAACAAATGTCTCCCTA
SYMS33 F TGCACTAACATGGTTGATGT 54 (GAAG)3
086 282E minus 90 Hypothetical proteinSORBIDRAFT 03g046450
SYMS33 R GGTGATTGTAAGGGTCATCTT
SYMS34 F GTTAATGGTGGTTCCGTTC 53 (GGC)6
088 4E minus 20 Predicted uncharacterizedprotein LOC101783547
SYMS34 R ATTATCAGCGCAGAGACATCSYMS35 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)
4075 100E minus 21 Preprotein translocase
SYMS35 R GTGTAGAACTGGAGCATTGAG
SYMS36 F GGACTGTACAAGGACGACAG 53 (GCT)4
070 114E minus 41 Protein transport proteinSec61 beta subunit
SYMS36 R TCTGCTTTCTTGGATATGGTA
SYMS37 F AAGAAGGATGCAAAGAAGAAG 54 (GAT)4
090 308E minus 81 Hypothetical proteinSORBIDRAFT 03g046450
SYMS37 R AGGCTTAGTAACAGCAGGTTTSYMS38 F AAGAAGGATGCAAAGAAGAAG 56 (AGA)
4086 900E minus 37 Hypothetical protein
SYMS38 R AGGCTTAGTAACAGCAGGTTTSYMS39 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 114E minus 41 Protein transport protein
SYMS39 R TCTGCTTTCTTGGATATGGTASYMS40 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 125E minus 40 Preprotein translocase
SYMS40 R TCTGCTTTCTTGGATATGGTA
SYMS41 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 962E minus 42 Protein transport proteinSec61 beta subunit
SYMS41 R TCTGCTTTCTTGGATATGGTASYMS42 F CCAAAGAGATCTTGCAGACTA mdash (ATG)
4mdash 178E minus 53 Jasmonate-induced protein
SYMS42 R CCCAACACAACAACCAATSYMS43 F CCACACAAGCAAGAAATAAAC mdash (GGT)
4mdash 857E minus 74 Dirigent-like protein
SYMS43 R TCGAACACTATGGTAAAGGTG
SYMS44 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41 Homeodomain-liketranscription factor
SYMS44 R TCTGCTTTCTTGGATATGGTASYMS45 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4069 986E minus 42 Protein transport protein
Genetics Research International 5
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS45 R GACTCTGCTTTCTTGGATATG
SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4
090 182E minus 44 Hypothetical proteinSORBIDRAFT 09g006220
SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)
3077 100E minus 21 Preprotein translocase Sec
SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)
6090 400E minus 20 TPA hypothetical protein
SYMS48 R CGTAAATATGAACGAGGTCAG
SYMS49 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein
SYMS49 R TCTGCTTTCTTGGATATGGTA
SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10
mdash 679E minus 13 TPA seed maturationprotein
SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)
4mdash 122E minus 41 Hypothetical protein
SYMS51 R ATTGTCACTTGCTATCCATTT
SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4
mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit
SYMS52 R GTAGATACCGAGCACACCAG
SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3
087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein
SYMS53 R GGATAGACTGAAATCTGCTCA
SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5
mdash 413E minus 08 Protein transport proteinSEC31
SYMS54 R GGAGGTGGAACTTCCTGA
SYMS55 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS55 R TCTGCTTTCTTGGATATGGTA
SYMS56 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)
6mdash 784E minus 27 Caltractin
SYMS57 R ATCAAAGAGATCAAAGGCTTC
SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6
074 597E minus 66Zinc finger A20 and AN1domains-containingprotein
SYMS58 R TAGGCTGCACAACAATAGTCT
SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6
080 402E minus 65 Predicted reticulon-likeprotein B1
SYMS59 R CAAGTACTCCAGCAGAGATGT
SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5
mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein
SYMS60 R TGTCACTAACACGAATCACAA
SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5
079 414E minus 57 Actin-depolymerizingfactor 3
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
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[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
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Molecular Biology International
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Signal TransductionJournal of
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Genetics Research International 3
FreeTree and TreeView software [18 19] The polymorphicinformation content (PIC) values were calculated for eachprimer by using the online resource of PIC Calculator (httpwwwlivacuksimkempsjpichtml)
26 Informative Assessment of Functional EST-SSR Markersbetween Bulks Plantmaterials were used as F2mapping pop-ulation comprising 209 genotypes of the sugarcane cultivarswhichwere developed from cross betweenCoS 91230 (ParentCoS 775 times Co 1148) with CoS 8436 (Parent MS 6847 timesCo 1148) from September to March (2010-2011) Groupingof genotypes was done according to their stem diameter(contrasting high and low stem diameter genotypes) intotwo sets DNA extractions were carried out from both setsand equal quantities of genomic DNA from 10 extreme highstem diameter and 10 extreme low stem diameter genotypeswere pooled into two bulks PCR amplification was donein both bulks with newly developed EST-SSR primers forinformativemarkers identifications through bulk segregationanalysis (BSA) [20]
3 Results and Discussion
31 Mining of Microsatellites in EST Sequences and SSRsCharacterization Total 10000 EST sequences related toSaccharum spp were examined from NCBI for the simplesequence repeat (SSR) identification and characterizationusing computational approach Prior to the marker decipher-ing sequence assembly was performed and 6201 (4201 kb)nonredundant sequences were detected comprising 1752contigs and 4449 singlets wherein 406 SSRs were identifiedwith 360 perfect SSRs and 37 sequences containing morethan 1 SSR and 30 SSRs in compound formation There-fore computational and experimental approach to ascertainmicrosatellites in EST libraries from public database (NCBI)turned to be very cost effective and reduces time and labourbesides expense of own libraries development EST-SSRs area more preferable DNA marker in the variety of geneticanalysis and found to be more conserved as present in thetranscribed region of the genome These were found to bemore transferable across the taxonomic boundaries and couldbe evaluated as most informative markers for variety ofgenomics applications [10 21] These are more adapted inplants comparative genetic analysis for gene identificationgene mapping marker-assisted-selection transferability andgenetic diversity [7 22ndash24] Also a variety of studies havebeen reported on sugarcane using EST-SSR markers fordesired genetic analysis [8 13 25 26]
The frequency of SSR in EST sequences was 64 includ-ing all the repeats except mononucleotide repeats This resultis comparatively higher compared to previous studies onsugarcane [8 27ndash29] Contrary to this Singh et al [13]reported higher frequency (93) in sugarcane Kumpatlaand Mukhopadhyay [30] also observed high range (265 to1062) of SSR frequency in different plant species In generalabout 5 of ESTs contained SSR which has been reportedin many plant species [31] These variations in microsatellitefrequency could be attributed to the ldquosearch criteriardquo usedtype of SSRmotif size of sequence data and the mining tools
used [24 32] In otherwords the density of themicrosatelliteswas one SSR per 1045 kb which is closely comparable toearlier studies in sugarcane with densities 1 SSR109 kb [8]and 19 kb SSR [13]
Analysis revealed that trinucleotide repeats (6674)were found to be more frequent followed by di- (2610)tetra- (467) penta- (15) and hexanucleotide (12)repeats Our observation of high frequency of trinu-cleotide repeats is in agreement with previous reports onsugarcane [8 13 27ndash29 33] Several other studies havealso represented high frequency of trinucleotide repeatsin different plant species [24 31 34ndash36] A total of 33different types of motifs were identified of which fourbelonged to dinucleotide eight belonged to trinucleotidestwelve belonged to tetranucleotide five belonged to pen-tanucleotide and two belonged to hexanucleotide repeats(Figure 1) We observed that motifs AGCT and ATATwere more frequent in dinucleotide repeat followed bymotifs CCGCGGAGCCTGAGGCCT andACGCGT intrinucleotide repeat motif AAAGCTTT in tetranucleotiderepeats motif ACAGGCCTGT in pentanucleotide repeatsand AACACCGGTGTT in hexanucleotide repeats Thepresence of motif CCGCGG was also observed in sugarcaneby different authors [13 27] Kantety et al [37] also reportedCCGCGG motif as most abundant in wheat and SorghumSimilarly both Lawson and Zhang [38] and Da Maia etal [39] also observed abundance of motif CCGCGG indifferent member of the grass family Victoria et al [35] alsodecoded motif CCGCGG in the lower plants (C reinhardtiiand P patens) Thus this predominance of CCGCGG motiffrequency has been related to a high GC-content [5] Somemotifs which are responsible for making unusual DNAfolding structure (hairpin formed bipartite triplex formedand simple loop folding) also have effect on gene expressionsand regulationsmechanism namely CCTAGG CCGGGCGGATTC and GAATTC motifs [40 41] Moreover thepresence of trinucleotide repeats in the coding region formeda distinct group and encoded amino acid tracts within thepeptide [42] We also observed predictable twenty differenttypes of amino acids including stop codon Alanine arginineglycine proline and serine were most frequent (Figure 2)This is in agreement with previous studies that reported ondifferent plant species [11 35 43]
32 Expressed Sequence Tags Annotation and PrimersDevelopment All EST sequences having SSRswere examinedby functional annotation (blastn blastx and gene ontology)After-effect sixty-three ESTs having SSRs were successfullyidentified on the basis of their involvement in the variousmetabolic processes (Figure 3) After-effect sixty-threeEST-SSRs primer pairs were designed for polymorphicnature cross transferability bulk segregation analysis andgenetic diversity in the test plants (Table 1) These selectedEST-SSRs comprised all types of repeat motifs (excludingmononucleotide repeat) and among trinucleotide repeatsthey were highly frequent with GCTCGA TCCAGGand GGTCCA repeat motifs Similarly Sharma et al [44]also used functional annotation pipelines for the moreprominent molecular markers development related to gene
4 Genetics Research International
Table 1 Details of selected 63 EST-SSR primer pairs used for cross transferability genetic diversity and bulks segregation analysis
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS28 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
081 939E minus 42 Protein transport proteinSec61 beta
SYMS28 R GTGTAGAACTGGAGCATTGAG
SYMS29 F GGGCAAGCAAGAAACCAC 52 (TCC)4
091 162E minus 24 Protein translation factorSUI1
SYMS29 R GAAGAGGTCAACCAAGAACTCSYMS30 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4086 100E minus 21 Preprotein translocase Sec
SYMS30 R GTGTAGAACTGGAGCATTGAG
SYMS31 F GAAGCTCCCAAGCTGCTA 53 (AGCT)3
076 200E minus 12 Predicted uncharacterizedprotein
SYMS31 R CCTACAGGAAAGATTTTAGGG
SYMS32 F GTCTCTTCTCCAGTTCTCCTT 55 (TGCG)4
084 246E minus 63Predictedactin-depolymerizingfactor
SYMS32 R GCTCAACAAATGTCTCCCTA
SYMS33 F TGCACTAACATGGTTGATGT 54 (GAAG)3
086 282E minus 90 Hypothetical proteinSORBIDRAFT 03g046450
SYMS33 R GGTGATTGTAAGGGTCATCTT
SYMS34 F GTTAATGGTGGTTCCGTTC 53 (GGC)6
088 4E minus 20 Predicted uncharacterizedprotein LOC101783547
SYMS34 R ATTATCAGCGCAGAGACATCSYMS35 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)
4075 100E minus 21 Preprotein translocase
SYMS35 R GTGTAGAACTGGAGCATTGAG
SYMS36 F GGACTGTACAAGGACGACAG 53 (GCT)4
070 114E minus 41 Protein transport proteinSec61 beta subunit
SYMS36 R TCTGCTTTCTTGGATATGGTA
SYMS37 F AAGAAGGATGCAAAGAAGAAG 54 (GAT)4
090 308E minus 81 Hypothetical proteinSORBIDRAFT 03g046450
SYMS37 R AGGCTTAGTAACAGCAGGTTTSYMS38 F AAGAAGGATGCAAAGAAGAAG 56 (AGA)
4086 900E minus 37 Hypothetical protein
SYMS38 R AGGCTTAGTAACAGCAGGTTTSYMS39 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 114E minus 41 Protein transport protein
SYMS39 R TCTGCTTTCTTGGATATGGTASYMS40 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 125E minus 40 Preprotein translocase
SYMS40 R TCTGCTTTCTTGGATATGGTA
SYMS41 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 962E minus 42 Protein transport proteinSec61 beta subunit
SYMS41 R TCTGCTTTCTTGGATATGGTASYMS42 F CCAAAGAGATCTTGCAGACTA mdash (ATG)
4mdash 178E minus 53 Jasmonate-induced protein
SYMS42 R CCCAACACAACAACCAATSYMS43 F CCACACAAGCAAGAAATAAAC mdash (GGT)
4mdash 857E minus 74 Dirigent-like protein
SYMS43 R TCGAACACTATGGTAAAGGTG
SYMS44 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41 Homeodomain-liketranscription factor
SYMS44 R TCTGCTTTCTTGGATATGGTASYMS45 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4069 986E minus 42 Protein transport protein
Genetics Research International 5
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS45 R GACTCTGCTTTCTTGGATATG
SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4
090 182E minus 44 Hypothetical proteinSORBIDRAFT 09g006220
SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)
3077 100E minus 21 Preprotein translocase Sec
SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)
6090 400E minus 20 TPA hypothetical protein
SYMS48 R CGTAAATATGAACGAGGTCAG
SYMS49 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein
SYMS49 R TCTGCTTTCTTGGATATGGTA
SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10
mdash 679E minus 13 TPA seed maturationprotein
SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)
4mdash 122E minus 41 Hypothetical protein
SYMS51 R ATTGTCACTTGCTATCCATTT
SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4
mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit
SYMS52 R GTAGATACCGAGCACACCAG
SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3
087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein
SYMS53 R GGATAGACTGAAATCTGCTCA
SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5
mdash 413E minus 08 Protein transport proteinSEC31
SYMS54 R GGAGGTGGAACTTCCTGA
SYMS55 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS55 R TCTGCTTTCTTGGATATGGTA
SYMS56 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)
6mdash 784E minus 27 Caltractin
SYMS57 R ATCAAAGAGATCAAAGGCTTC
SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6
074 597E minus 66Zinc finger A20 and AN1domains-containingprotein
SYMS58 R TAGGCTGCACAACAATAGTCT
SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6
080 402E minus 65 Predicted reticulon-likeprotein B1
SYMS59 R CAAGTACTCCAGCAGAGATGT
SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5
mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein
SYMS60 R TGTCACTAACACGAATCACAA
SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5
079 414E minus 57 Actin-depolymerizingfactor 3
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
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[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
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[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
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Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
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4 Genetics Research International
Table 1 Details of selected 63 EST-SSR primer pairs used for cross transferability genetic diversity and bulks segregation analysis
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS28 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
081 939E minus 42 Protein transport proteinSec61 beta
SYMS28 R GTGTAGAACTGGAGCATTGAG
SYMS29 F GGGCAAGCAAGAAACCAC 52 (TCC)4
091 162E minus 24 Protein translation factorSUI1
SYMS29 R GAAGAGGTCAACCAAGAACTCSYMS30 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4086 100E minus 21 Preprotein translocase Sec
SYMS30 R GTGTAGAACTGGAGCATTGAG
SYMS31 F GAAGCTCCCAAGCTGCTA 53 (AGCT)3
076 200E minus 12 Predicted uncharacterizedprotein
SYMS31 R CCTACAGGAAAGATTTTAGGG
SYMS32 F GTCTCTTCTCCAGTTCTCCTT 55 (TGCG)4
084 246E minus 63Predictedactin-depolymerizingfactor
SYMS32 R GCTCAACAAATGTCTCCCTA
SYMS33 F TGCACTAACATGGTTGATGT 54 (GAAG)3
086 282E minus 90 Hypothetical proteinSORBIDRAFT 03g046450
SYMS33 R GGTGATTGTAAGGGTCATCTT
SYMS34 F GTTAATGGTGGTTCCGTTC 53 (GGC)6
088 4E minus 20 Predicted uncharacterizedprotein LOC101783547
SYMS34 R ATTATCAGCGCAGAGACATCSYMS35 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)
4075 100E minus 21 Preprotein translocase
SYMS35 R GTGTAGAACTGGAGCATTGAG
SYMS36 F GGACTGTACAAGGACGACAG 53 (GCT)4
070 114E minus 41 Protein transport proteinSec61 beta subunit
SYMS36 R TCTGCTTTCTTGGATATGGTA
SYMS37 F AAGAAGGATGCAAAGAAGAAG 54 (GAT)4
090 308E minus 81 Hypothetical proteinSORBIDRAFT 03g046450
SYMS37 R AGGCTTAGTAACAGCAGGTTTSYMS38 F AAGAAGGATGCAAAGAAGAAG 56 (AGA)
4086 900E minus 37 Hypothetical protein
SYMS38 R AGGCTTAGTAACAGCAGGTTTSYMS39 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 114E minus 41 Protein transport protein
SYMS39 R TCTGCTTTCTTGGATATGGTASYMS40 F GGACTGTACAAGGACGACAG mdash (GCT)
4mdash 125E minus 40 Preprotein translocase
SYMS40 R TCTGCTTTCTTGGATATGGTA
SYMS41 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 962E minus 42 Protein transport proteinSec61 beta subunit
SYMS41 R TCTGCTTTCTTGGATATGGTASYMS42 F CCAAAGAGATCTTGCAGACTA mdash (ATG)
4mdash 178E minus 53 Jasmonate-induced protein
SYMS42 R CCCAACACAACAACCAATSYMS43 F CCACACAAGCAAGAAATAAAC mdash (GGT)
4mdash 857E minus 74 Dirigent-like protein
SYMS43 R TCGAACACTATGGTAAAGGTG
SYMS44 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41 Homeodomain-liketranscription factor
SYMS44 R TCTGCTTTCTTGGATATGGTASYMS45 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)
4069 986E minus 42 Protein transport protein
Genetics Research International 5
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS45 R GACTCTGCTTTCTTGGATATG
SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4
090 182E minus 44 Hypothetical proteinSORBIDRAFT 09g006220
SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)
3077 100E minus 21 Preprotein translocase Sec
SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)
6090 400E minus 20 TPA hypothetical protein
SYMS48 R CGTAAATATGAACGAGGTCAG
SYMS49 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein
SYMS49 R TCTGCTTTCTTGGATATGGTA
SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10
mdash 679E minus 13 TPA seed maturationprotein
SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)
4mdash 122E minus 41 Hypothetical protein
SYMS51 R ATTGTCACTTGCTATCCATTT
SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4
mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit
SYMS52 R GTAGATACCGAGCACACCAG
SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3
087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein
SYMS53 R GGATAGACTGAAATCTGCTCA
SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5
mdash 413E minus 08 Protein transport proteinSEC31
SYMS54 R GGAGGTGGAACTTCCTGA
SYMS55 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS55 R TCTGCTTTCTTGGATATGGTA
SYMS56 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)
6mdash 784E minus 27 Caltractin
SYMS57 R ATCAAAGAGATCAAAGGCTTC
SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6
074 597E minus 66Zinc finger A20 and AN1domains-containingprotein
SYMS58 R TAGGCTGCACAACAATAGTCT
SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6
080 402E minus 65 Predicted reticulon-likeprotein B1
SYMS59 R CAAGTACTCCAGCAGAGATGT
SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5
mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein
SYMS60 R TGTCACTAACACGAATCACAA
SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5
079 414E minus 57 Actin-depolymerizingfactor 3
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Genetics Research International 5
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS45 R GACTCTGCTTTCTTGGATATG
SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4
090 182E minus 44 Hypothetical proteinSORBIDRAFT 09g006220
SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)
3077 100E minus 21 Preprotein translocase Sec
SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)
6090 400E minus 20 TPA hypothetical protein
SYMS48 R CGTAAATATGAACGAGGTCAG
SYMS49 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein
SYMS49 R TCTGCTTTCTTGGATATGGTA
SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10
mdash 679E minus 13 TPA seed maturationprotein
SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)
4mdash 122E minus 41 Hypothetical protein
SYMS51 R ATTGTCACTTGCTATCCATTT
SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4
mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit
SYMS52 R GTAGATACCGAGCACACCAG
SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3
087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein
SYMS53 R GGATAGACTGAAATCTGCTCA
SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5
mdash 413E minus 08 Protein transport proteinSEC31
SYMS54 R GGAGGTGGAACTTCCTGA
SYMS55 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS55 R TCTGCTTTCTTGGATATGGTA
SYMS56 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)
6mdash 784E minus 27 Caltractin
SYMS57 R ATCAAAGAGATCAAAGGCTTC
SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6
074 597E minus 66Zinc finger A20 and AN1domains-containingprotein
SYMS58 R TAGGCTGCACAACAATAGTCT
SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6
080 402E minus 65 Predicted reticulon-likeprotein B1
SYMS59 R CAAGTACTCCAGCAGAGATGT
SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5
mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein
SYMS60 R TGTCACTAACACGAATCACAA
SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5
079 414E minus 57 Actin-depolymerizingfactor 3
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
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[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
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BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Genetics Research International
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Volume 2014
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International Journal of
Microbiology
6 Genetics Research International
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS61 R CAGTCACAAAGTCGAAATCAT
SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3
085 440E minus 66 Truncated alcoholdehydrogenase
SYMS62 R CCAATCTTGACATCCTTGAC
SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4
067 311E minus 31 Hypothetical proteinSORBIDRAFT 08g002800
SYMS63 R CAGCTTCACTCATGAATTTTT
SYMS64 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 108E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)
4051 342E minus 74 Dirigent-like protein
SYMS65 R AACACTATGGTCAAGGTGGTA
SYMS66 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)4
058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS66 R GAAATCGCTCTATAAGGTTCC
SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5
090 425E minus 83 Hypothetical proteinSORBIDRAFT 03g005100
SYMS67 R GTTAAGAGGCTTCCAAAGAAC
SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5
200E minus 55Putativeubiquitin-conjugatingenzyme family
SYMS68 R GTGGCTTGTTTGGATATTCTT
SYMS69 F GGACTGTACAAGGACGACAG 54 (GCT)4
079 928E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)
4077 158E minus 73 Putative dirigent protein
SYMS70 R AACACTATGGTCAAGGTGGTA
SYMS71 F GGACTGTACAAGGACGACAG mdash (GCT)4
mdash 986E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS71 R TCTGCTTTCTTGGATATGGTA
SYMS72 F CCCTCTCCCTGCTCTTTC 55 (TCC)4
089 436E minus 57 Actin-depolymerizingfactor 3
SYMS72 R CAGTCACAAAGTCGAAATCAT
SYMS73 F GGACTGTACAAGGACGACAG 55 (GCT)4
083 109E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)
4088 951E minus 42 Preprotein translocase Sec
SYMS74 R TCTGCTTTCTTGGATATGGTA
SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3
093 178E minus 68 TPA general regulatoryfactor 1
SYMS75 R CGGTAGTCCTTGATGAGTGT
SYMS76 F GGACTGTACAAGGACGACAG 52 (GCT)4
078 479E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS76 R TCTGCTTTCTTGGATATGGTA
SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3
093 834E minus 70 Hypothetical proteinSORBIDRAFT 10g030160
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Genetics Research International 7
Table 1 Continued
Serialnumber Type Primer sequence Annealing
temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)
SYMS77 R AAGTTGATTCACCCTCATTCT
SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3
092 104E minus 41Translocon-associatedprotein alpha subunitprecursor
SYMS78 R AAGTTGATTCACCCTCATTCT
SYMS79 F GGACTGTACAAGGACGACAG 53 (GCT)4
091 104E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS79 R TCTGCTTTCTTGGATATGGTA
SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6
087 225E minus 59Predictedubiquitin-conjugatingenzyme E2
SYMS80 R ATTGCTGTTGATATTTGGATG
SYMS81 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
087 801E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS81 R GTGTAGAACTGGAGCATTGAG
SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4
090 112E minus 30 Glycine-rich RNA-bindingprotein 2
SYMS82 R GGCTATAGTCACCACGGTAG
SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4
087 939E minus 42Protein transport proteinSec61 subunit beta-likeisoform
SYMS83 R GACTCTGCTTTCTTGGATATG
SYMS84 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
075 114E minus 41Protein transport proteinSec61 subunit beta-likeisoform
SYMS84 R AATCGCTCTATAAGGTTCCTC
SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6
mdash 114E minus 51Protein transport proteinSec61 subunit beta-likeisoform
SYMS85 R CAAACCTCATAAAGAGTGCAG
SYMS86 F GGGCAAGCAAGAAACCAC 54 (TCC)4
093 116E minus 28 TPA translation initiationfactor 1
SYMS86 R CGTACATGAACGTAGTCCTTT
SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4
mdash 119E minus 41 Protein transport proteinSec61 beta subunit
SYMS87 R AATCGCTCTATAAGGTTCCTC
SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4
mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970
SYMS88 R CACCAAGTACTCATCCATCAT
SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4
091 964E minus 60 Predicted NACdomain-containing protein
SYMS89 R ATTTATAGGTTGGCACCAGAG
SYMS90 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4
085 100E minus 22Protein transport proteinSec61 subunit beta-likeisoform
SYMS90 R GTGTAGAACTGGAGCATTGAG
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
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[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
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[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
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Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
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Genetics Research International
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Advances in
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Nucleic AcidsJournal of
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Stem CellsInternational
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 Genetics Research International
020406080
100120140
ACG
TAG
CT
ATA
TCG
CG
AAC
GTT
AAG
CTT
AAT
ATT
ACC
GG
TAC
GC
GT
ACT
AGT
AGC
CTG
AGG
CCT
ATC
ATG
CCG
CG
GA
AAG
CTT
TA
AAT
ATT
TA
AGG
CCT
TA
ATG
ATT
CA
ATT
AAT
TAC
GC
CGTG
ACTC
AG
TGAG
CCC
TGG
AGCG
CG
CTAG
GC
CCTG
AGG
GC
CCT
CCG
GC
CGG
AA
AAG
CTT
TTA
ATAT
ATA
TTAC
AGG
CCT
GT
ACCT
CAG
GTG
AGCG
GC
CGCT
AAC
ACC
GG
TGTT
AGCA
GG
CCT
GCT
Types of motifs
Freq
uenc
y
Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity
010203040506070
Ala
Arg
Asn Asp Cy
s
Gln
Glu Gly His Ile Leu
Lys
Met
Phe
Pro
Val
Types of amino acids
Freq
uenc
y
Ser
Stop Ty
r
Thr
Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs
transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)
33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications
produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]
34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
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Enzyme Research
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International Journal of
Microbiology
Genetics Research International 9
Primary metabolic process
Cellular metabolic process
Response to stress
Macromolecule metabolic process
Cellular response to stimulus
Transport
Response to other organisms
Response to biotic stimulus
Establishment of protein localization
Response to abiotic stimulus
Immune response
Response to chemical stimulus
Regulation of biological process
Cellular component organizationCellular component organization or
biogenesis at cellular levelNitrogen compound metabolic process
Small molecule metabolic process
Catabolic process
Oxidation-reduction process
Establishment of localization in cell
Biosynthetic process
Anatomical structure morphogenesis
Actin filament-based process
Cell cycle process
Vesicle-mediated transport
Post-embryonic development
Interspecies interaction between organisms
Developmental process involved in reproduction
Cellular developmental process
Activation of immune response
Cell growth
Regulation of biological quality
Immune effector processModification of morphology or
physiology of other organismSecondary metabolic process
Embryo development
Cellular localization
Seed germination
Multicellular organismal reproductive processAnatomical structure formation
involved in morphogenesisTransmembrane transport
Dormancy process
Cellular component movement
Cell division
Response to endogenous stimulus
01 02 03 04 05 06 07 08 09 1000
00 01 02 03 04 05 06 07 08 09 10
Biological_process of GO Iv3
(a)
Figure 3 Continued
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
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[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
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Advances in
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
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Enzyme Research
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International Journal of
Microbiology
10 Genetics Research International
00 01 02 03 04 05 06 07 08 09 10
Cellular_component of GO Iv3
090807 1000 0604030201 05
External encapsulating structureEnvelope
Vesicle Cell-cell junction
Membrane part Ribonucleoprotein complex
Protein complex Organelle membrane
Non-membrane-bounded organelle Intracellular organelle part
Membrane Membrane-bounded organelle
Intracellular organelle Intracellular part
(b)
0807 09 1005 06030201 0400
00 01 02 03 04 05 06 07 08 09 10
Molecular_function of GO Iv3
transcription factor activitySequence-specific DNA binding
Transmembrane transporter activityTetrapyrrole binding
Oxidoreductase activity Ligase activity
Nucleotide binding Hydrolase activity
Ion binding Protein binding
Nucleic acid binding Substrate-specific transporter activity
(c)
Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)
51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the
genome are expected to be more conserved and more trans-ferable across taxa
35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Genetics Research International 11Ta
ble2Detailsof
crosstransferabilityof
42ES
T-SSRmarkersin
twentygeno
typesb
elon
ging
tocerealsa
ndSaccharum
related
genera
andSaccharum
speciesa
ndtheirc
ultiv
arsLanes1
to20
representn
umbero
fbands
prod
uced
inwheatm
aizebarleyric
epearlm
illetoatSorghum
NarengaScle
rosta
chyaEria
nthu
sMiscanthusB
andjermasin
Hita
mG
unjer
a51NG56N
58
CoS
92423CoS
88230UP9530C
oS91230andCoS
8436respectively
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
284
56
12
30
25
53
23
23
31
32
395
SY29
63
59
312
35
126
77
87
76
76
68
100
SY30
10
40
07
05
66
33
43
23
23
23
80SY
310
00
00
00
23
21
12
22
02
20
050
SY32
10
01
02
02
43
13
00
10
00
00
55SY
330
02
23
53
76
46
55
52
30
23
385
SY34
00
00
00
00
00
03
25
45
55
99
85SY
350
00
10
40
52
10
01
11
22
12
165
SY36
00
00
00
00
30
05
22
20
20
02
35SY
379
75
66
55
912
1011
88
88
67
65
6100
SY38
00
03
02
07
81
00
22
12
00
30
50SY
450
00
00
20
51
01
21
10
00
00
035
SY46
01
44
10
04
40
10
00
00
00
00
35SY
470
13
32
00
10
00
00
00
00
00
025
SY48
00
00
53
08
120
53
32
13
22
00
60SY
530
00
11
33
129
21
12
21
32
13
185
SY58
00
01
10
32
10
00
00
00
00
00
25SY
590
00
00
00
40
33
00
10
00
00
020
SY61
03
75
40
34
56
34
15
35
55
55
90SY
620
00
10
20
45
32
20
00
00
00
035
SY63
00
00
00
01
14
16
11
10
10
21
55SY
650
00
00
00
00
11
10
12
32
22
150
SY66
00
00
00
01
00
03
12
00
32
21
40SY
671
25
63
30
25
63
20
02
00
00
060
SY69
15
20
10
03
12
11
11
12
10
32
80SY
701
00
10
10
52
00
10
02
03
30
045
SY72
10
20
00
01
45
08
58
13
64
68
70SY
730
02
01
20
22
01
31
41
03
00
360
SY74
21
10
11
31
32
14
15
10
30
02
80SY
751
02
33
00
06
39
76
76
04
60
065
SY76
00
00
02
01
21
40
00
10
00
00
30SY
772
56
68
43
96
93
04
43
43
02
085
SY78
22
56
46
45
76
410
910
32
87
59
100
SY79
00
00
10
03
52
05
17
72
10
06
55SY
801
10
40
10
45
62
54
55
35
45
585
SY81
00
01
26
06
68
73
34
33
33
32
80SY
820
010
31
43
91
16
54
35
34
43
290
SY83
12
06
23
42
72
13
23
13
32
33
95
SY84
00
01
23
00
20
03
31
03
33
33
60
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
12 Genetics Research International
Table2Con
tinued
Snu
mberlane
12
34
56
78
910
1112
1314
1516
1718
1920
Prim
erpo
lymorph
ism(
)SY
861
56
32
54
58
116
53
115
127
67
10100
SY89
96
54
17
37
43
104
45
73
47
35
100
SY90
50
32
52
34
34
04
36
44
23
64
90Av
erageo
ftransfe
rability
2722
2722
4722
4667
3611
5556
2611
8833
9889
7111
600
7333
5556
7556
550
5056
5889
5111
5278
600
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Genetics Research International 13
2019181716151413121110987654321M
50bp
100 bp
200 bp300bp400bp
600bp1050bp1350bp
Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436
CoS 92423
51NG56
Bandjermasin Hitam
GunjeraCoS 88230UP 9530
CoS 8436CoS 91230
BarleyRice
Maize
Wheat
Pearl milletSorghum Erianthus
NarengaMiscanthus
N58
Oat
Sclerostachya
III
III
Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software
most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem
36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks
SYMS69SYMS66SYMS53SYMS45SYMS30
SYMS89SYMS83SYMS82SYMS81SYMS72
Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis
of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
14 Genetics Research International
4 Conclusion
The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities
References
[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006
[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987
[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998
[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005
[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993
[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008
[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014
[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010
[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014
[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007
[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010
[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011
[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013
[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008
[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012
[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994
[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001
[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999
[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996
[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991
[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007
[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Genetics Research International 15
[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009
[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013
[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003
[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005
[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001
[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004
[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009
[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005
[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005
[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007
[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012
[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010
[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011
[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014
[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002
[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006
[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009
[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001
[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002
[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005
[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011
[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009
[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013
[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005
[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002
[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011
[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004
[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005
[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009
[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009
[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012
[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
16 Genetics Research International
scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011
[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005
[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006
[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014
[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993
[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994
[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013
[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003
[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004
[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996
[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997
[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001
[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006
[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007
[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010
[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant
analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology