AGRICULTURAL RESEARCH COMMUNICATION CENTREwww.arccjournals.com/www.legumeresearch.in

*Corresponding author’s e-mail: drsusheelsharma@rediffmail.com

Diversity analysis of pea genotypes using RAPD markersBharti Thakur, Susheel Sharma*, Isha Sharma, Prachi Sharma, Deepika and Sajad Majeed Zargar

Division of Plant Pathology,Sher-e-Kashmir University of Agricultural Sciences and Technology-Jammu-180 009, Jammu and Kashmir, India.Received: 23-03-2016 Accepted: 11-03-2017 DOI: 10.18805/LR-3709

ABSTRACTGenetic diversity among 54 genotypes of field and garden pea of India were studied using 30 RAPD primers. A total of 168amplicons were scored, of which 154 were polymorphic revealing 89.3% of polymophism. Out of 30 RAPD primerstested, 22 primers produced clear and reproducible bands. The number of products generated by these primers ranged from04 to 14 with primer OPU-17 giving maximum (14) and primer OPC-08 giving minimum number of amplicons (04).Genetic similarity estimates based on the binomial data using Jaccard’s coefficient ranged from 0.34 (Kaza-2/IC-218991)to 0.89 (IC-209118/IC-209123) with an average similarity index of 0.54 exhibiting considerable diversity among the peagenotypes studied. The dendrogram showed clear pattern of clustering according to the source of germplasm. This studyindicated that Himalayan region germplasm exhibited higher diversity.

Key words: Genetic diversity, Pisum sativum, RAPD.

INTRODUCTIONAmong legumes, pea (Pisum sativum L.; 2n = 14),

belonging to family Leguminosae, is an important coolseason crop with a rich history in genetic research datingback to the classical work by the father of genetics, GregorJ. Mendel. The relatively narrow gene pool and use of asmall number of varieties as parents by competing breedingprograms have led to low genetic diversity among peacultivars (Baranger et al. 2004). The efficiency of breedingprogramme in crop species depends on the availability ofgenetic base for that crop as more the diversity moresuccessful is the breeding (Singh et al.2011). Many of thewild forms of peas grow wildly or are grown as subsistencepulse crop since ages in the Himalayan regions of Jammuand Kashmir (J&K) and Himachal Pradesh (H.P.) and thusthese areas are thought to be rich in primitive cultivated formsof field pea (Kapila et al. 2011). The consumers also have aspecial preference for pea from hilly regions due to itscharacteristic flavor, good quality and sweetness (Kumar etal., 2015). Molecular markers can overcome the difficultyover morphological markers in analyzing genetic divergenceof increasing number of reference germplasm collections.Many studies in peas in the past have been conducted onassessment of genetic diversity among sub-sets of germplasmof different regions, use groups and seasons usingmorphological and/or molecular markers including RAPD,ISSR, AFLP and SSR markers (Simioniuc et al. 2002 andTar’an et al. 2005). RAPD markers are important not onlyfor the characterization of the germplasm but can also beused to assess the effects of selection over time and to helpin the development of cross breeding programmes since this

process allows the study of the genetic diversity of theexisting germplasm (Nicese et al., 1998). A foremostadvantage of RAPD markers over some other DNA basedmarkers is that they necessitate no prior sequenceinformation, and no prior knowledge regarding any particulargene in a target taxon. This technique is helpful to developgenotype-specific banding patterns important for cultivaridentification (Gaafar and Saker, 2006). In the present study,RAPD markers were used to characterize field and gardenpea germplasm for the identification of potent diverse geneticresources of pea.MATERIALS AND METHODSCollection of seed material:Fifty four genotypes of Pisumwere used for molecular characterization. The seed materialwas collected from National Bureau of Plant GeneticResources,(NBPGR), New Delhi and Shimla (HP), ChanderShekher Azad University and Technology (CSAUT), Kanpur,CSK Himachal Pradesh Krishi Vishvavidyalaya (CSKHPKV), Palampur, upper Himalayan tracts of HimachalPradesh and Leh-Ladakh regions of J&K (Table 1). The seedmaterial was multiplied for a season to check for the geneticpurity of the accessions through phenotypic characters.DNA extraction and PCR amplification: Genomic DNAwas isolated from the young leaves of accessions by adoptingthe procedure outlined by Doyle and Doyle (1990). Thequantity and quality of DNA was estimated by mySPECTM

and gel electrophoresis. The stock DNA was diluted to 20–25 ng/l for further PCR analysis. A set of 22 RAPD primerswas used for molecular characterization (Table 2). PCR wasconducted in 25 l of reaction volume containing 2 l (20 –

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Table 1: Details of the germplasm collected for genetic diversity analysis.Codes Genotype Source of Procurement Codes Genotype Source of Procurement1 IC-209118 NBPGR, New Delhi 28 EC-381866 NBPGR, Shimla2 IC-209123 NBPGR, New Delhi 29 EC-598535 NBPGR, Shimla3 IC-209125 NBPGR, New DelhiI 30 EC-598538 NBPGR, Shimla4 IC-209126 NBPGR, New Delhi 31 EC-598655 NBPGR, Shimla5 IC-218984 NBPGR, New Delhi 32 EC-598704 NBPGR, Shimla6 IC-218986 NBPGR, New Delhi 33 EC-598729 NBPGR, Shimla7 IC-218987 NBPGR, New Delhi 34 EC-598757 NBPGR, Shimla8 IC-218991 NBPGR, New Delhi 35 EC-598816 NBPGR, Shimla9 IC-218993 NBPGR, New Delhi 36 EC-598878 NBPGR, Shimla10 IC-218997 NBPGR, New Delhi 37 Azad P-1 CSAUT, Kanpur11 IC-219002 NBPGR, New Delhi 38 Azad P-2 CSAUT, Kanpur12 IC-219008 NBPGR, New Delhi 39 Azad P-3 CSAUT, Kanpur13 IC-219009 NBPGR, New Delhi 40 Azad P-4 CSAUT, Kanpur14 IC-219010 NBPGR, New Delhi 41 Azad P-5 CSAUT, Kanpur15 IC-279113 NBPGR, New Delhi 42 Arkel CSK HPKV, Palampur16 IC-279217 NBPGR, New Delhi 43 Bonnevillae CSK HPKV, Palampur17 IC-311065 NBPGR, New Delhi 44 Lincoln CSK HPKV, Palampur18 IC-326345 NBPGR, New Delhi 45 Prakash SKUAST-Jammu19 IC-328514 NBPGR, New Delhi 46 Rachna SKUAST-Jammu20 IC-328701 NBPGR, New Delhi 47 Kalpa Upper Himalayas of HP21 IC-329410 NBPGR, New Delhi 48 Kalpa Bold Upper Himalayas of HP22 IC-329586 NBPGR, New Delhi 49 Kaza-1 Upper Himalayas of HP23 IC-208366 NBPGR, Shimla 50 Kaza-2 Upper Himalayas of HP24 IC-208378 NBPGR, Shimla 51 Hammar Leh-Ladakh region of J&K25 IC-218988 NBPGR, Shimla 52 Diskit Nubra Leh-Ladakh region of J&K26 IC-267142 NBPGR, Shimla 53 Partapur Nubra Leh-Ladakh region of J&K27 IC-278261 NBPGR, Shimla 54 Sumoor Nubra Leh-Ladakh region of J&k

25 ng/l) of genomic DNA, 2.50 l 10× PCR buffer, 1.5 lof MgCl2 (25 mM), 1 l dNTPs (10 mM each, Thermo), 1.0l primer (10 pM), 0.2 l of Taq DNA polymerase (5 U/l)and 16.8 l of sterilized distilled water. Amplifications werecarried out using a DNA thermal cycler (peqSTAR) byoptimizing the protocol, with the following parameters: initialdenaturation for 3 min at 95ºC followed by 38 cycles,denaturation of 1 min at 95ºC, annealing of 1 min at 36ºC or37ºC extension at 72ºC for 1.30 min and final extension at72ºC for 10 min. Amplified PCR products were resolved byelectrophoresison 1.5 % agarose gel.Statistical analysis: Reproducible RAPD products weremanually scored for band presence (1) or absence (0) foreach accession and a binary qualitative data matrix wasconstructed. The potential of the markers for estimatinggenetic variability among pea genotypes was examined byprimer banding characteristics like total number of bands(NB), number of polymorphic bands (NPB) percentage ofpolymorphic bands (PPB), polymorphic information content(PIC), resolving power (Rp) and marker index (MI) (Table 3).

The PIC value for each locus was calculated usingformula (Roldan-Ruiz et al. 2000); PIC = 2fi (1 - fi), wherePIC is the polymorphic information content, fi is thefrequency of the ampliûed fragments and 1 - fi is thefrequency of non-ampliûed fragments. The frequency was

calculated as the ratio between the number of ampliûedfragments at each locus and the total number of accessions(excluding missing data). The PIC of each primer wascalculated using the average PIC value from all loci of eachprimer. The resolving power (RP) of each primer wascalculated as (Prevost and Wilkinson 1999); RP = Ib, whereIb represents the informative fragments. The Ib can berepresented on a scale of 0/1 by the following formula; Ib =1 - (2 9 |0.5 - pi|), where pi is the proportion of accessionscontaining the ith band. The data matrix was converted intogenetic similarity matrix using Jaccard coeficient andNTSYS-PC 2.0 (Rohlf 1998).RESULTS AND DISCUSSION

Out of 30 RAPD primers tested, 22 primers (Table2) produced clear and reproducible amplicons (Figure 1).The number of products generated by these primers werefound to range from 04 to 14 with primer OPU-17 givingthe maximum (14) and primer OPC-08 giving the minimumnumber of amplicons (04). A total of 168 amplicons rangingfrom 200 to 5000 bp were produced out of which 154(89.32%) were polymorphic. The percentage ofpolymorphism ranged from 25% for OPC-08 to 100% forOPA-07, OPA-10, OPA-11, OPB-10, OPC-18, OPD-07,OPD-18, OPE-02, OPE-16, OPO-02, OPU-16 and OPX-01. Three unique bands were also recognized out of 154polymorphic amplicons.

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Table 2: List of 22 RAPD primers along with their sequencesS. No. Primer Primer Sequence S. No. Primer Primer Sequence1 OPA-02 TGCCGAGCTG 12 OPD-18 GAGAGCCAAC2 OPA-07 GAAACGGGTG 13 OPE-02 GGTGCGGGAA3 OPA-09 GGGTAACGCC 14 OPE-03 CCAGATGCAC4 OPA-10 GTGATCGCAG 15 OPE-16 GGTGACTGTG5 OPA-11 CAATCGCCGT 16 OPN-06 GAGACGCACA6 OPB-10 CTGCTGGGAC 17 OPO-02 ACGTAGCGTC7 OPC-02 GTGAGGCGTC 18 OPU-08 GGCGAAGGTT8 OPC-08 TGGACCGGTG 19 OPU-16 CTGCGCTGGA9 OPC-09 CTCACCGTCC 20 OPU-17 ACCTGGGGAG10 OPC-18 TGAGTGGGTG 21 OPX-01 CTGGGCACGA11 OPD-07 TTGGCACGGG 22 OPY-15 AGTCGCCCTT

Table 3: Various parameters depicting informativeness of 22 RAPD primersPrimer NB NPB NMB NUB PPB PIC MI RpOPA-02 6 5 0 1 83.3 0.164 0.683 1.330OPA-07 7 7 0 0 100 0.297 2.079 2.968OPA-09 7 5 2 0 71.42 0.259 0.925 2.518OPA-10 6 6 0 0 100 0.169 1.014 1.182OPA-11 10 10 0 0 100 0.406 4.060 6.486OPB-10 7 7 0 0 100 0.417 2.919 4.960OPC-02 6 5 1 0 83.33 0.351 1.462 3.572OPC-08 4 1 3 0 25 0.009 0.002 0.038OPC-09 8 6 2 0 75 0.206 0.927 2.374OPC-18 10 10 0 0 100 0.403 4.030 6.290OPD-07 5 5 0 0 100 0.281 1.405 2.072OPD-18 10 10 0 0 100 0.270 2.700 3.994OPE-02 10 10 0 0 100 0.397 3.970 6.334OPE-03 6 5 1 0 83.33 0.269 1.120 2.408OPE-16 6 6 0 0 100 0.346 2.076 2.964OPN-06 6 5 1 0 83.33 0.245 1.020 1.854OPO-02 9 9 0 0 100 0.379 3.411 5.110OPU-08 5 4 1 0 80 0.310 0.992 2.516OPU-16 7 7 0 0 100 0.268 1.876 2.588OPU-17 14 13 0 1 92.85 0.318 3.830 6.624OPX-01 11 11 0 0 100 0.471 5.181 8.730OPY-15 8 7 0 1 87.5 0.310 1.898 3.628Average 7.63 7 0.5 0.136 89.32 0.2975 2.162 3.66NB = Number of bands PPB = polymorphic percentage of bandsNPB = Number of polymorphic bands PIC = Polymorphic information contentNMB = Number of monomorphic bands Rp = Resolving powerMI = Marker index

The discriminatory power of RAPDs wasdetermined by calculating parameters viz., percentage ofpolymorphic bands (PPB), polymorphic information content(PIC), marker index (MI) and resolving power (Rp). HighPIC value of 0.471 (OPX-01) and low PIC value of 0.009(OPC-08), with an average value of PIC per primer 0.29was observed. The maximum PIC was calculated for OPX-01 (0.471) followed by OPA-11 (0.406), OPC-18 (0.403)and OPE-02 (0.397). The highest MI was observed with theprimer OPX-01 (5.181) and lowest in the primer OPC-08(0.002), with an average MI of 2.16 per primer. The resolvingpower (Rp) is a parameter that indicates the discriminatorypotential of the primers chosen. The highest Rp value was

observed for primer OPX-01 (8.73) and lowest for primerOPC-08 (0.038), with an average Rp of 3.66 per primer.Ten RAPD primers (OPA-11, OPB-10, OPC-02, OPC-18,OPD-18, OPE-02, OPO-02, OPU-17, OPX-01 and OPY-15) exhibited high (>3.00) resolving power.

Genetic similarity estimates based on the binomialdata using Jaccard’s coefficient ranged from 0.34 (Kaza-2/IC-218991) to 0.89 (IC-209118/IC-209123) with an averagesimilarity index of 0.54 exhibiting considerable diversityamong the pea genotypes studied. All the wild collectionshave shown less degree of similarity with the cultivatedvarieties and other collections displaying substantialdiversity. Whereas high similarity estimates were revealed

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P l a t e A

P l a t e A

P l a t e B

P l a t e B

Figure 1: RAPD banding profile of OPE-16 (Plate A) and OPO-02 (Plate B)

within the cultivated varieties thus showing less diversity.The similarity estimates were then used to execute clusteranalysis using UPGMA. Germplasm under study wasgrouped into four main clusters A, B, C and D, comprisingof 18, 18, 11 and 7 genotypes, respectively (Figure 2). Thedendrogram showed clear pattern of clustering according tothe source of the germplasm.

Many aspects viz., importance of the crop,increasing demand for protein-rich raw materials as a proteinsource (Santalla et al. 2001), relatively narrow gene pool(Heath and Hevvlethwate, 1985) and the heavy use of a smallnumber of varieties as parents by competing breedingprograms have led to low genetic diversity among peacultivars (Baranger et al. 2004) and thus steering plantbreeders for continuous exploration of novel resources andestablishing genetic relationships among the germplasm. Inthe present study on diversity analysis, comprising of 54genotypes, a total of 168 amplicons were scored, of which154 were polymorphic revealing 89.3% of polymophism.Simioniuc et al. (2002); Tar’an et al. (2005); Choudhury etal.(2007) and Kapila et al. (2011) have reported 55.7%, 44%,

74.8% and 72.3% PPB in their studies on peas using RAPD,respectively. PIC value ranges from zero for monomorphicmarkers to 0.5 for markers that are present in 50% of theplants and absent in the other 50% (Ruiz et al. 2000). Thevalues of PIC in our study ranged from 0.009 to 0.471.RAPDs viz., OPX-01 (0.471), OPA-11 (0.406), OPC-18(0.403) and OPE-02 (0.397) with high PIC value canpotentially be used by other researchers. To determine thegeneral usefulness of the system of markers used, the MI(marker index) for each RAPD primer was calculated. Thehighest MI was observed with the primer OPX-01 (5.181)and lowest in the primer OPC-08 (0.002), with an averageMI of 2.16 per primer. However, Choudhury et al. (2007)have reported more MI (4.787) in important varieties of peain India. The highest Rp was observed with the primer OPX-01 (8.73) and the lowest with the primer OPC-08 (0.038),with an average Rp of 3.66 per primer. Ten RAPD primers(OPA-11, OPB-10, OPC-02, OPC-18, OPD-18, OPE-02,OPO-02, OPU-17, OPX-01 and OPY-15) exhibited high(>3.00) resolving power, therefore, suggesting their potentialto be used for resolving/identifying these pea genotypes from

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mixed populations. Similarly, Kapila et al. (2011) have alsoreported five RAPD markers with Rp > 3.00 as potentialmarkers.

Genetic similarity estimates based on the binomialdata using Jaccard’s coefficient ranged from 0.34 (Kaza-2/IC-218991) to 0.89 (IC-209118/IC-209123) with an averagesimilarity index of 0.54 exhibiting considerable diversityamong the pea genotypes studied. However, Choudhury etal. (2007) have reported higher (0.71) similarity indexsuggesting moderate diversity in their material.Correspondingly, Kapila et al. (2011) have reported theJacccard’s coefficients ranging from 0.58-0.85. Similarly,genetic relationships have also been established by variousworkers in peas viz., Simioniuc et al. (2002); Baranger etal.(2004) and Smykal et al.(2008). All the wild collectionshave shown less degree of similarity with the cultivated

varieties and other collections and thus can be used tobroaden the gene pool. Whereas, high similarity estimateswere revealed within the cultivated varieties depicting theirnarrow genepool. The similarity estimates were then usedto execute cluster analysis using UPGMA. Fifity fourgenotypes were grouped into four main clusters A, B, C andD. Cluster ‘A’ comprised of 18 genotypes all belonging toindigenous collections (IC). Cluster ‘B’ comprised ofindigenous and exotic collections. Cluster ‘C’ consisted ofall the commercial varieties showing less genetic difference.Cluster analysis was unable to completely separate Rachnaand Prakash (field pea varieties) from garden pea varietieswhich is in agreement with earlier studies in pea (Tar’an etal.2005; Smykal et al.2008;Kapila et al.2011) but is incontrast to the studies of Simioniuc et al.(2002) and Barangeret al. (2004). This might be due to reason that the latterhave

Fig-2: Dendrogram depicting the genetic relationship among 54 genotypes of Pisum based on Jaccard’s similarity coefficient

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also included seed storage protein and isozyme data in theirstudies. Cluster ‘D’comprised of all the wild accessionscollected from high hills of J&K and HP. Kalpa bold hasbeen clustered in commercial variety group ‘C’ with Lincolnrather than wild group ‘D’. Kalpa Bold was collected fromKalpa region of Himachal Pradesh and has wrinkled and boldseeds as compared to Kalpa accession which is smooth andsmall seeded. This clustering might have occurred becauseof inadvertent spread of a commercial variety (may beLincoln) and it possibly have started growing wildly in theseareas. Clustering of all the commercial varieties in one singleclade signifies the selection pressure imposed for breedingvarious economical traits. As expected, the dendrogramshowed clear pattern of clustering according to the sourceof the germplasm. Thus, from the present experiment it canbe inferred that RAPD based approach is capable of revealing

sufficient variability among the germplasm under study.RAPDs can serve as a useful approach to identify peagenotypes used in the study. The five primers viz., OPX-01,OPE-02, OPA-11, OPE-10 and OPC-18 were remarkablyinformative and can be used by other workers in their study.Majority of pea collections displayed considerable diversityfrom commercial varieties of garden and field peas andtherefore can potentially be used in pea breeding programmesto broaden the genetic base of existing varieties as putativeassurance against unpredicted biotic and abiotic stresses.ACKNOWLEDGEMENT

The funding received from SERB, Department ofScience and Technology, New Delhi under SB/YS/LS-182/2013 is gratefully acknowledged. The authors are alsothankful to NBPGR, New Delhi for providing thegermplasm.

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