Research Article A Simple and Efficient Method to Isolate ...

Research ArticleA Simple and Efficient Method to Isolate LTR Sequences ofPlant Retrotransposon

Da-Long Guo1 Xiao-Gai Hou2 and Xi Zhang2

1 College of Forestry Henan University of Science and Technology Luoyang Henan 471003 China2 College of Agriculture Henan University of Science and Technology Luoyang 471003 China

Correspondence should be addressed to Da-Long Guo guodl2005126com and Xiao-Gai Hou hxg382126com

Received 6 February 2014 Revised 22 March 2014 Accepted 9 April 2014 Published 6 May 2014

Academic Editor Jozef Anne

Copyright copy 2014 Da-Long Guo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Retrotransposons (RTNs) have important roles in the formation of plant genome size structure and evolution Ubiquitousdistributions abundant copy numbers high heterogeneities and insertional polymorphisms of RTNs have made them as excellentsources for molecular markers development However the wide application of RTNs-based molecular markers is restricted by thescarcity of the LTR (long terminal repeat) sequences information A new simple and efficient method to isolate LTR sequences ofRTNs was presented based on the degenerate RNase H nested primers and PPT (polypurine tract) primer of RTNs in tree peonyThis method combined the characteristics and advantages of high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR)annealing control primer (ACP) system and suppression PCR method Nineteen LTR sequences were isolated using this newmethod in tree peony and the applicability of the LTR sequences based markers was validated by further SSAP analysis The resultsshowed that the new method is simple of low-cost and highly efficient which is just conducted by three rounds of PCR and doesnot need any restriction enzymes and adapters much less the hybridizations This new method is rapid economical and cost- andtime-saving which could be easily used to isolate LTR sequences of RTNs

1 Introduction

Retrotransposons (RTNs) are the most abundant and wide-distributed mobile genetic elements in eukaryotic genomesand they inserted into the host genome via an RNA inter-mediate [1] RTNs can be divided into two major groupsLTR (long terminal repeat) and non-LTR retrotransposonsaccording to the presence or absence of LTR [2] LTR-RTNsare ubiquitous in the plant kingdom and present in highcopy numbers and constitute major parts of plant genomeswhich can comprise 40ndash90 of the genome as a whole[2] RTNs have important roles in the formation of plantgenome size structure and evolution [2ndash4] They have beenemployed as an efficient tool in gene cloning gene expressionand phylogenetic analysis [5 6] Ubiquitous distributionsabundant copy numbers high heterogeneities and inser-tional polymorphisms both within and between plant LTRretrotransposons have made them as excellent sources formolecular markers development [6 7] and obtained more

attractions than other conventional markers due to theirspecific characteristics [7] The molecular markers based onthe RTNs have been developed constantly such as SSAP [8]RBIP [9] iPBS [10] IRAP and REMAP [11] and have shownlarge superiority over other conventional molecular markers[6 12 13] These RTN-based markers revealed insertionpolymorphism between RTNs and other elements due totheir different primer design principles [7] Usually SSAPshows more polymorphism and more codominance thanAFLP [7] and it is more informative for studying geneticdiversity in tomato and pepper than SSR and AFLP [12] Amajor disadvantage of all the methods described above isthe need for LTR sequence information of RTNs to designspecies-specific primers [7] especially for SSAP

RTNs have many conserved motifs such as sequence ofreverse transcriptase (RT) [3] RNase H [14] primer bindingsite (PBS) [10] and polypurine tract (PPT) [15] LTR-RTNsshare a unique structural feature Two long terminal repeats(LTRs) are longer than 100 bp and play a key role in their

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 658473 8 pageshttpdxdoiorg1011552014658473

2 BioMed Research International

transposition [2] PBS is adjacent to the 51015840-LTR and used toprime the reverse transcriptase-catalyzed synthesis of minus-strand cDNA [10] PPT is located upstream of the 31015840-LTR andis a site of plus-strand synthesis initiation [2 16] Anotherimportant element of RTNs RNase H is responsible forthe degradation of the RNA template in the DNA-RNAhybrid The specific structural features of LTR-RTNs havebeen described by Wicker et al [17]

Pearce et al [14] firstly reported a novel technique forrapid isolation of plant Ty1-copia terminal repeat sequencesof RTNs based on the consensus amino acids of RNase Hmotif After that various isolation methods of LTR-RTNshave been developed based on the conserved primers [10 14]and genome walking methods [15 18ndash21] These methodshave different principles and efficiencies but some of themeither need probe hybridization or need enzyme digestionand adapter ligationThese lead to the tedious procedures andinfluence the isolation efficiency Compared with the existingmethods for isolation of LTR sequence the key factor is theefficiency of chromosome walking which is used to cloneunknown LTR region based on the conserved RNase H orPPT motif of RTNs [15 18ndash22]

In this study a new easy fast and efficient isolationmethod of RTN-LTRs was proposed and validated in treepeonyThis method combined the characteristics and advan-tages of high-efficiency thermal asymmetric interlaced PCR(hiTAIL-PCR) [23] annealing control primer (ACP) system[24] and suppression PCR [22 25] method It is simple low-cost and highly efficient which is just conducted throughthree rounds of PCR and does not need any restrictionenzymes and adapters much less the hybridizations

2 Materials and Methods

21 Materials and DNA Extraction Tree peony (Paeonia suf-fruticosa Andrews) cultivar ldquoLuoyanghongrdquo was used toisolate the LTR sequenceThe fresh leaveswere collected fromthe Luoyang National GenBank of Tree Peony China TotalDNAwas extracted from the fully expanded true leaves usingthe CTAB method [26]The material information used forSSAP analysis is shown in Table 1

22 PCR Amplification The components of PCR reactionand PCR program of three rounds were presented in Tables2 and 3 respectively The nested RNase H primers used inthis study were according to that of Pearce et al [14] TheACP primers were followed as that of Hwang et al [24] TheUP (universal primer) primer was designed based on theprimers of suppression PCR [25] PPT primer was designedto be degenerate primer based on the sequence characteristicsof PPT of RTNs [16] The corresponding primers sequencesare shown in Table 4 PCR products from the third roundof PCR were cloned into the PMD-18T vector using the TACloning Kit (TaKaRa Dalian China) The ligation productswere transformed into DH5120572 competent cell and positiveclones were sequenced by Sun Biotech Co (Beijing China)

23 Sequence Analysis The nature of cloned sequences wasconfirmed by performing similarity searches with knownRTNs sequences from other plants in NCBI database usingBLASTN BLASTX and TBLASTX algorithms with thedefault parameters GenomicDNA sequences were depositedin the GenBank databases Multiple DNA sequence align-ments were carried out using Lasergene 80 with MegAlignmodule by Jotun Hein method The LTR and PPT segmentsof the sequence were identified by comparing with the knownstructural characteristics of LTR and PPT regions of RTNs[14 15]

24 SSAP Analysis SSAP amplification was performed asdescribed by Bousios et al [19] SSAP analysis was alsocarried out twice for each primer pair in order to check theconsistency and reproducibility of the SSAP markers Thesequences of SSAP adapters and adapter primers were thesame as Vos et al [27] RTN primers were designed basedon the isolated LTR sequences according to the method ofWaugh et al [8] Selective amplification was conducted witha RTNs primer in combination with eitherMse I + 3 or EcoRI + 3 [27] The resulting bands were detected using the silverstaining protocol following the method of Bassam et al [28]

3 Results and Discussion

31 The Primer Design Principle of the NewMethod The suc-cess of PCR amplification relies on the binding specificitythat is a primer anneals to its target sequences There-fore it is important to optimize this molecular interaction[29] In order to improve the isolation efficiency of LTRsequence the method of high-efficiency TAIL-PCR (hiTAIL-PCR) presented by Liu and Chen [23] was firstly consideredwhich allows efficient amplification of large unknown targetsequences [23] The longer AD (LAD) primers of hiTAIL-PCR are 33 or 34 nucleotides which contained six or sevendegenerate nucleotides and four fixed nucleotides at the31015840 ends The arbitrary 31015840 four-base sites have a moderatefrequency (on average 256 bp at a time) to anchor the LADsto the unknown target regions Three nested specific primersets based on the known sequences were used to conductgenome walking in combination with the LAD primers Thespecific PCR products were amplified through the thermalasymmetric interlaced PCR programs and subsequent PCRreactions In order to employ hiTAIL-PCR in the newmethod the nested specific primers in the original hiTAIL-PCR were replaced by two nested RNase H primers [14] andone PPT degenerate primer based on the conserved motifof RNase H and PPT of RTNs At the same time it is a keyfactor for the success of PCR amplification that the primerscould bind to its target sequences or not at the period ofannealing Therefore the optimization of primer structureis very crucial for PCR amplification The primer annealingtemperature determines that the primer completely bindsto the template or partially mismatches the template in oneor several bases Consequently the adjustment of primerannealing temperature could improve the combination speci-ficity of primers and template Hwang et al [24] designed

BioMed Research International 3

Table 1 Characteristics of tree peony genotypes used for SSAP analysis

Code Cultivar (species) Flower form Cultivar group Color1 Luoyanghong Rose form Zhongyuan Purple2 Yinhongqiaoyu Chrysanthemum form Zhongyuan Red3 Lantianyu Crown form Zhongyuan Blue4 Doulv Globular form Zhongyuan Green5 Er Qiao Rose form Zhongyuan Bicolor6 Fengdan Single form Jiangnan White7 P rockii Single form The wild White8 P ludlowii Single form The wild Yellow

Table 2 The components of three PCR rounds respectively

Component First PCRamplification

Second PCRamplification

Third PCRamplification

Template DNA 50ndash100 ng1000 times dilution ofPCR products from

the first PCR

1000 times dilution ofPCR products fromthe second PCR

Buffer (TaKaRa) 1x 1x 1xMg2+ 25mmolsdotLminus1 25mmolsdotLminus1 25mmolsdotLminus1

dNTPs 02mmolsdotLminus1 02mmolsdotLminus1 02mmolsdotLminus1

RNase H1 primer 04 120583molsdotLminus1 mdash mdashRNase H2 primer mdash 04 120583molsdotLminus1 mdashACP primer 08 120583molsdotLminus1 mdash mdashPPT primer mdash mdash 04 120583molsdotLminus1

Universal primer UP mdash 04 120583molsdotLminus1 04 120583molsdotLminus1

Taq polymerase 10U 10U 10UTotal volume 10 120583L 20 120583L 20 120583L

the annealing control primer (ACP) in order to enhance thespecificity of PCR amplification ACP primer is composedof three parts a polydeoxyinosine [poly(dI)] linker betweenthe 31015840 end target core sequence and the 51015840 end nontargetuniversal sequenceThe poly(dI) linker prevents annealing ofthe 51015840 end nontarget sequence to the template and facilitatesprimer hybridization at the 31015840 end to the target sequence atspecific temperatures resulting in a dramatic improvementof annealing specificity [24]

In order to further improve the specificity the universalprimer (UP primer) of suppression PCR was used whichtakes advantage of the priority of intrachain annealing thaninterchain ones When there are inverted repeat sequencesin both ends of PCR products (ie UP primer) the endsof the nontarget individual DNA strands will form ldquopan-handlerdquo structures (stem-loop structures) following everydenaturation step These structures would affect the bond ofthe primers and template are more stable than the primer-template hybrid and therefore will suppress exponentialamplification [22 25] While a distal gene-specific primerextends a DNA strand through the UP primer site theextension product will contain the UP primer sequence onlyon one end and thus cannot form the ldquopanhandlerdquo structurePCR amplification can then proceed normally [25]

The following are the summaries of primer design of thisnew method

(A) The binding sites of the target region in the unknownregionswere created by the degenerate primers (LAD)of hiTAIL-PCR However the 31015840 end specific geneprimer of original ACP primer was replaced by theLAD primer of hiTAIL-PCR

(B) The specificity of PCR amplification was improved bythe suppression PCR through the introduction of UPprimer And it has got rid of the need of digestion andligation by thisUPprimer acted as the adapter primerof suppression PCR and the original 51015840 end of ACPprimer The different LAD primers shared a commonsequence in the 51015840 half that is UP primer

(C) The binding specificity of the primer with the tem-plate was controlled by ACP primers which consistedof UP primer at the 51015840 end intermediate [poly(dI)]linker and LADprimers of hiTAIL-PCR at the 31015840 end

(D) The conserved primers of RNase H and PPT motifof RTNs replaced the gene-specific primers in thehiTAIL-PCR

(E) The proportion of nontarget products was reducedby increasing the dilution ratio of the template in thenext PCR amplification

32 General Outline of the Technique In total three roundsof nested PCRs were employed in this study to improve the


Table 3 The programme parameters of three rounds of PCR

Phase Step Cycle conditions Cycles

First PCR round

1 94∘C for 5min 12 94∘C for 50 s 60∘C for 1min and 72∘C for 2min 53 94∘C for 50 s 45∘C for 30 s and 72∘C for 2min 14 94∘C for 50 s 55∘C for 30 s and 72∘C for 2min 255 72∘C for 8min 1

Second PCR round1 94∘C for 5min 12 94∘C for 50 s 55∘C for 30 s and 72∘C for 2min 303 72∘C for 10min 1

Third PCR round1 94∘C for 5min 12 94∘C for 1min 55∘C for 30 s and 72∘C for 2min 303 72∘C for 10min 1

Table 4 The sequences of primers used in this study

Primers Sequence

Nested primer RNase H1 MGNACNAARCAYATHGARNase H2 GCNGAYATNYTNACNAA

ACP primer

ACP 1 TGTAGCGTGAAGACGACAGAA IIIII VNVNNNGGAAACP 2 TGTAGCGTGAAGACGACAGAA IIIII BNBNNNGGTTACP 3 TGTAGCGTGAAGACGACAGAA IIIII HNVNNNCCACACP 4 TGTAGCGTGAAGACGACAGAA IIIII CAATGGCTACCACACP 5 TGTAGCGTGAAGACGACAGAA IIIII VVNVNNNCCAAACP 6 TGTAGCGTGAAGACGACAGAA IIIII BDNBNNNCGGT

UP primer UP TGTAGCGTGAAGACGACAGAAPPT primer RRRRRRRRRRRRRRRRNote the bold letters represent the nontarget universal sequences I represent deoxyinosineThemeaning of degenerate base B (CGT) D (AGT)H (ACT)R (AG) N (AGCT) and V (ACG)

specificity Firstly the reaction was conducted in order tofacilitate the RNase H1 primer to hybridize to the conservedRNase H domain of the template under the stringent condi-tions For the ACP primer 31015840 end degenerate primer couldanneal to the template and 51015840 end could not anneal at thehigh annealing temperature Therefore ACP primer formeda vesicular structure due to the hybridization failure of lowannealing [poly(dI)] to the template in this condition

In fact three kinds of different concentrations of PCRproducts were obtained after the first step as shown inFigure 1 Product A is amplified from the combinations ofRNase H1 primer and different ACP-LAD primers ProductB is just from ACP-LAD primer by itself while Product Conly resulted from RNase H1 primer Among these productsProduct A is the exact target products Product C also seemsas one of the target products in some extent but it is very fewand nearly could not be amplified due to the long distance oftwoRTNs Product B is not the target products But Product Bonly with ACP-LAD primer would not be amplified becausethey could not hybridize to the template under the highannealing temperature In addition nontarget products fromnonspecific priming by the ACP-LAD primer alone if anyare diluted and cannot be amplified in the following PCRsusing the nested specific primers Therefore the target coresequences rise up and nontarget products are very few

After 5 cycles of linear amplification of target sequencesprimed by RNase H1 primer and different ACP-LAD primerwhich increases the copy numbers of the target moleculesa single cycle with a low annealing temperature (45∘C) iscarried out The higher degree of the primer degeneracy andthe low annealing temperature allow the LAD primer to bindto the target sequence with a higher probability thus moretarget LTR sequence regions were efficiently created [23]Then the target sequences are amplified steadily at moderateannealing temperature (55∘C) to increase their amounts inthe following steps

Then the second round of PCR was followed withthe 1000 times diluted PCR products as the template andamplified with the nested RNase H 2 primer and UP primerThe nonspecific products primed only by UP primer tend toform a stem-loop structure due to their complementary endswhich suppresses their amplification in the next amplification[25] Thus it decreased the nonspecific products On theother hand new nontarget products by UP primer alonecannot be generated and amplified to visible levels from suchdiluted (approximately 1000-fold) templates in the secondPCR

At last the diluted PCR products of the second roundwere amplified for another nested PCR with PPT primerand UP primer The specific products were obtained through


LTRIR IR

Gag

3998400

3998400

5998400

5998400

5998400

3998400

3998400

5998400

INT RTGenomic DNA

First amplification

RNase H1 primer

RNase H1 primer RNase H1 primer

Target products

Product A

+

+

Nested primer RNase H2

Nested primer PPT

Universal primer UP

Universal primer UP

Second amplification

No products

Formation

Stem-loop

Could not be amplified

RNase H

RNase H

RNase H

RNase H

RNase H1

RNase H2

PPT

PPT PPT

PPT

LTR

RNase H PPT LTR

IRNontarget products

Product B

Product C

PPT

PPT

PPT

Sequencing the products

Third amplification

LTR

LTR LTR

LTR

LTR

LTR

ACP primer

PR

3998400

3998400

5998400

5998400

3998400

5998400

Figure 1 The schematic of the new method to isolate the LTR sequence of retrotransposons

2000 bp

500bp

100 bp

Figure 2 Amplification results from three rounds of PCR inLuoyanghong using the new method It represented three rounds ofPCR results from left to right M DL 2000 marker 1ndash6 ACP primer1ndash6

this hierarchical PCR reaction and nontarget products werefarthest suppressed

33 Isolation of Tree Peony LTR Sequences The LTR se-quences of tree peony RTNs were isolated with the newlydevelopedmethod in this studyThe representative tree peonycultivar in China ldquoLuoyanghongrdquo (Paeonia suffruticosa) wasused to perform the nested PCR The amplification resultsof three rounds of PCR were shown in Figure 2 A smearof bands was usually showed by RNase H1 and one of theACP-LAD primers in the first round of PCR (Figure 2)which contained a large number of randomly amplifiedproducts which resulted from tree peony genomic DNA Insome cases the first round of amplified products were notdetectable on the agarose gels (data were not shown) buttarget products could still be obtained in the next PCR Insome circumstances the second amplification would alsoproduce DNA smears But the differential shift between thefirst and second products on agarose gels is a good indicatorof the product specificity In this study the products resultingfrom the second round of PCR have some extent of specificity(Figure 2) When the third round of PCR was conducted the

specific products as a specific strong fragmentwere producedwhich indicated the success

A total of 22 independent clones were randomly selectedand sequenced After the exclusion of repeated sequences19 sequences possessed the expected PPT primer and UPprimer giving an average success rate of 8636 Thesesequences have been deposited in GenBank and the acces-sion numbers are from GenBank KC519444 to GenBankKC519464 Few duplicates were obtained and so it is likelythat many more new LTRs of RTNs could be obtained by thecharacterization of more subclonesThis is much higher thanthat (50ndash70) of the original TAIL-PCRprocedure [23] Allsix LAD primers worked well in our tests

Putative LTR sequence was firstly identified usingTEClass [30] which is a software to identify different typeof RTNs by universal structural features shared by LTRs-RTNs And the characteristics of internal RTNs regions werealso compared to the known structures of RTNs that is thepolypurine tract site with the conserved AGGGGGAGmotif[31] is located immediately upstream of the right LTR [32]

These sequences were aligned using Lasergene 80 andthe alignment results were shown in Figure 3 A continuousGA nucleotide motif (AGGGGG) that is highly conservedin PPT motif was observed in all 19 sequences and it wasfollowed by the TGTA sequence which is the indication ofthe beginning of LTR sequences However not all identifiedLTRs began with the canonical sequences ldquoTGrdquo which hasalso been observed in the study of Galindo et al [15]Although these regions differed in length (9ndash16 bp) andsequence characteristics they all contained a conservedmotif51015840-AGGGG-31015840 [16] While the distance of the predicted PPTsfrom the 31015840-LTR start varied between 0 bp and 3 bp themedian of 1 bp indicated that theyweremostly located at theirexpected positions as well [33] These characteristics of PPT-LTR junction are also reported elsewhere [14 15] supporting


KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H

KC519447KC519448KC519449KC519450KC519451KC519452KC519453KC519454KC519455KC519456KC519457KC519458KC519459KC519460KC519461KC519462KC519463KC519464Consensus

5998400 3

998400

AGAAGGGGG--TA-CTGCTCGGTC-AGAAGGGGGG--TG-GAACCGGTGT-

AGAAGGGGGGG--TG-GTTGCGGTGT-AGAAGGGGGG--TG-GAACCGGTGT-

AGAAGGGGGCGG-TTG-TCGGTGGGGC-AGAAGGGGGGG-TTG-TCCGGTATCA-

AGAAGGGGG-TTG-CTGGCGGTCC-AGAAGGGGGG--TG-TTGGCGGTCG-AGAAGGGGG--TG-GGTGACGGTG-

AGAAGGGGGGAA--TG-GCTACCACCA-AGAAGGGGGGAA--TG-GCTACCCCAT-GGGGAAAGGGAA--TG-AGTAGGCCCG-AGAAAGGGGGAA--TG-GCTACCACAT-

GAAGAGGAGAGAAAAG-TTA-TGCTGATGCA-AGAAGGGGGG--TG-GAACCGGTGT-AGAAGGGGG--TG-GGTGACGGTG-

AGAAGGGAGAGGA-TTG-GTTGATTTTG-AGAAGGGGG--TA-CTGCTCAGTC-

AGAAGGGGGCGG-TTG-TCGGTGGGGC------- Poly Pu--TG------------------------------

(389 bp)-TTGTCATTTG-TTCTGTCGTCTTCACGCTACA(343 bp)-ATCGTTACGG-TTCTGCTGATTACCCCCACCA(383 bp)-TTGTGTGTGT-TTCTGTGGTTGAACTCGATGT(373 bp)-ATTGTTGCAG-TTCTGTCGTCTTCACGCTACA(372 bp)-GGGTTTGTTG-TTCTGTCGTCTTCACGCTACA(326 bp)-CTGACTCTTG-TTCTGAATTCCCTGAGTAGGT(437 bp)-ACCATGAGTG-TTCTGTCGTCTTCAAGCTACA(244 bp)-ATCGATCCTC-TTCATATGAATTGTATTTACT(392 bp)-TGGGTCTTGG-TTCTGTCGTCTTCACGCTACA(248 bp)-ATGAGCTATA-TTCTGTCGTCTTCACGCTACA(406 bp)-TTCGGTCCTA-TTCTGTCGTCTTCACGCTACA(290 bp)-CATTGCCCCC-TTCTGTCGTCTTCACGCTACA(266 bp)-GTCTTGTCCC-TTCTACCGGCGATGCGCACCT(495 bp)-CATTGCCCCC-TTCTGTCGTCTTCACGCTACA

-ATGGCAGCAG-TTCTGTCGTCTTCACGCTACA(369 bp)(369 bp)-TCAACGGCGG-TTCTCAGCACTGGTGGATCTT

-AGGTGGTATC-TTGTGAGCATGATTTGTGGTT(388 bp)(365 bp)-CACAGAATAA-TTCCAGTACTTGACTTGTCAT(345 bp)-ATGGCGGCGG-TTCCGTCGTCTGAATCTGGGT

TTCTGTCGTCTTCACGCTACA

Figure 3 Alignment of nucleotide sequences of polypurine tracts (PPT) and 31015840-LTR terminal sequences Intervening sequence is of the sizeindicated in brackets

500bp

140bp

20bp

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8MPSLTR3M-CAT PSLTR2M-CAG PSLTR4E-AGC

Figure 4 Comparison of SSAP profiles obtained with differentretrotransposon and adapter primers Each retrotransposon primerwas used in combination with the Mse I or EcoR I adapter primerEach set of eight lanes displays the reactions from the materials inTable 1 (from left to right) The primers of PSLTR 2 3 and 4 aredesigned based on the sequence of GenBank KC519450 GenBankKC519454 and GenBank KC519459 respectively

the reliability of the obtained LTR sequences herein LTRsequences are highly variable both in sequence characteristicsand in length These sequences showed a similarity of 40ndash998 with an average of 189 in nucleotide sequences

34 SSAP Analysis SSAP is a RTNs-based marker systemthat utilizes sequence-specific RTNs-derived primers in com-bination with AFLP adapter primers It is the most popular

RTNs-based molecular marker method at present [6 1321] A number of LTRs were isolated in this study In anattempt to evaluate the utility of these LTR sequences asmolecular markers we conducted a PCR survey to detect thepolymorphic bands in a set of diverse tree peony genotypeswhich include different tree peony species cultivar groupscolors and flower forms (Table 1) The results showedthat primers designed on these LTRs allowed to evidencehighly polymorphic SSAP fingerprints in Paeonia Exampleof SSAP silver staining result was shown in Figure 4 Ahigh quality profile displays abundant intense bands againsta low background A large number of clear bands with ahigh percentage of polymorphisms were produced whichwere corresponding to the diversities of the diverse materialsThese results showed that the SSAP marker system basedon the isolation of tree peony LTR sequences could be usedeffectively for exploring polymorphism among tree peonyvarieties

35 The Characteristics and Advantage of the New MethodThe method reported herein has several advantages overprevious studies on the isolation of LTR sequences [14 15 1819]

351 Low False Positive Kalendar et al [10] designed iPBSprimers and used them not only as molecular markers butalso as the isolation method of RTN-LTR sequences Theycould obtainmany sequences at one time however amajorityof these sequences may lack typical or complete RTN-LTRsequences [10 34] The high false positive is a main obstacleof TAIL-PCR due to its short random primer (10sim13 bp)which resulted in the nonspecific binding with template inlow annealing temperature The new method employed theimproved hiTAIL-PCR [23] which used the longer LAD


primer and in combination with ACP system which have a[poly(dI)] linker [24] So it makes the modified ACP primerscould specifically anneal to the template in a high annealingtemperature (60∘C) during the initial step

352 EasyApplication Thismethod is just based onPCRandagarose gel technique It directly aimed at the LTR region andhas removed the necessity of the procedure of hybridizationwashing screening and so forth which is used in themethod of Pearce et al [14] Compared with other isolationmethods the PCR-RAGE method [20] requires the enzymedigestion and other tedious procedures the SiteFinding-PCRmethod [18] also needs enzyme digestion and another sixgene-specific primers with two SiteFinder primers The newmethod did not need enzyme digestion or adapter ligation Itjust needs the PCR so it is very easy

353 High Repeatability The method of Pearce et al [14]needs several times of hybridization and washing Becausethe efficiency of hybridization andwashing is largely differenteach time so the results of different reiteration would bevariedwhich affected the repeatabilityThemethod of Zhao etal [18] needs to design six gene-specific primers so differentgene-specific primers had to be designed when every newLTR sequence was isolated This method just employs threenested primers and one special designing primer whichrandomly combined in the target region and has simplifiedthe heavy and complicated steps of hiTAIL-PCR to reduce thechance of error imported therefore the repeatability is highlyimproved due to the high annealing temperatureThe specificprimer in the original ACP system has been replaced by theLAD primers

354 Rapid and Economical All reactions of this methodcould be completed only in one day The target sequencecould be obtained in short time and does not need to excludethe false positive Otherwise the additional conductionswereneeded to identify the reliability of candidate sequence inother methods [14 20] This method could isolate enoughtarget sequences one time and could be applied to anotheramplification of unknown sequence in the vicinity of anyknown sequence except for the isolation of LTR sequences

4 Conclusion

A new PCR method to isolate the LTR sequences of RTNswas developed in tree peony This method combined theadvantages of hiTAIL-PCR suppression PCR and annealingcontrol primer (ACP) methods and reduced the nonspecificamplification by random degenerate primers in hiTAIL-PCRby suppression effects of suppression PCR and high annealingtemperature increasing of primers This method is rapideconomical and cost- and time-saving which could be easilyused to isolate LTR sequences of RTNs in other plants

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Natural Science Foundationof China (NSFC 31070620 31370697) Program for Scienceamp Technology Innovation Talents in Universities of HenanProvince (13HASTIT004) and key Technologies R amp DProgram of Hersquonan Scientific Committee (132102110029)

References

[1] M-A Grandbastien ldquoRetroelements in higher plantsrdquo Trendsin Genetics vol 8 no 3 pp 103ndash108 1992

[2] A Kumar and J L Bennetzen ldquoPlant retrotransposonsrdquoAnnualReview of Genetics vol 33 no 1 pp 479ndash532 1999

[3] A J Flavell E Dunbar R Anderson S R Pearce R Hart-ley and A Kumar ldquoTy1-copia group retrotransposons areubiquitous and heterogeneous in higher plantsrdquo Nucleic AcidsResearch vol 20 no 14 pp 3639ndash3644 1992

[4] D F Voytas M P Cummings A Konieczny F M Ausubel andS R Rodermel ldquocopia-Like retrotransposons are ubiquitousamong plantsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 89 no 15 pp 7124ndash71281992

[5] A Kumar and H Hirochika ldquoApplications of retrotransposonsas genetic tools in plant biologyrdquo Trends in Plant Science vol 6no 3 pp 127ndash134 2001

[6] A H Schulman A J Flavell E Paux and T H N Ellis ldquoTheapplication of LTR retrotransposons as molecular markers inplantsrdquoMethods inMolecular Biology vol 859 pp 115ndash153 2012

[7] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011

[8] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997

[9] A J Flavell M R Knox S R Pearce and T H N EllisldquoRetrotransposon-based insertion polymorphisms (RBIP) forhigh throughput marker analysisrdquoThe Plant Journal vol 16 no5 pp 643ndash650 1998

[10] R Kalendar K Antonius P Smykal and A H SchulmanldquoiPBS a universal method for DNA fingerprinting and retro-transposon isolationrdquoTheoretical and Applied Genetics vol 121no 8 pp 1419ndash1430 2010

[11] R Kalendar TGrobMRegina A Suoniemi andA SchulmanldquoIRAP and REMAP two new retrotransposon-based DNAfingerprinting techniquesrdquo Theoretical and Applied Geneticsvol 98 no 5 pp 704ndash711 1999

[12] S M Tam C Mhiri A Vogelaar M Kerkveld S R Pearceand M-A Grandbastien ldquoComparative analyses of geneticdiversities within tomato and pepper collections detected byretrotransposon-based SSAP AFLP and SSRrdquo Theoretical andApplied Genetics vol 110 no 5 pp 819ndash831 2005

[13] I Castro C DrsquoOnofrio J P Martın et al ldquoEffectiveness ofAFLPs and retrotransposon-based markers for the identifica-tion of Portuguese grapevine cultivars and clonesrdquo MolecularBiotechnology vol 52 no 1 pp 26ndash39 2012

[14] S R Pearce C Stuart-Rogers M R Knox A Kumar T H NEllis and A J Flavell ldquoRapid isolation of plant Ty1-copia group


retrotransposon LTR sequences for molecular marker studiesrdquoThe Plant Journal vol 19 no 6 pp 711ndash717 1999

[15] L M Galindo E Gaitan-Solıs P Baccam and J Tohme ldquoIso-lation and characterization of RNase LTR sequences of Ty1-copia retrotransposons in common bean (Phaseolus vulgarisL)rdquo Genome vol 47 no 1 pp 84ndash95 2004

[16] J Macas and P Neumann ldquoOgre elementsmdasha distinct group ofplant Ty3gypsy-like retrotransposonsrdquo Gene vol 390 no 1-2pp 108ndash116 2007

[17] T Wicker F Sabot A Hua-Van et al ldquoA unified classificationsystem for eukaryotic transposable elementsrdquo Nature ReviewsGenetics vol 8 no 12 pp 973ndash982 2007

[18] G Zhao Z Zhang H Sun H Li and H Dai ldquoIsolation of Ty1-copia-like retrotransposon sequences from the apple genomeby chromosome walking based on modified sitefinding-poly-merase chain reactionrdquo Acta Biochimica et Biophysica Sinicavol 39 no 9 pp 675ndash683 2007

[19] A Bousios I Saldana-Oyarzabal A G Valenzuela-Zapata CWood and S R Pearce ldquoIsolation and characterization of Ty1-copia retrotransposon sequences in the blue agave (Agave tequi-lana Weber var azul) and their development as SSAP markersfor phylogenetic analysisrdquo Plant Science vol 172 no 2 pp 291ndash298 2007

[20] L Natali T Giordani M Buti and A Cavallini ldquoIsolation ofTy1-copia putative LTR sequences and their use as a tool toanalyse genetic diversity in Olea europaeardquoMolecular Breedingvol 19 no 3 pp 255ndash265 2007

[21] X Du Q Zhang and Z Luo ldquoDevelopment of retrotransposonprimers and their utilization for germplasm identification inDiospyros spp (Ebenaceae)rdquo Tree Genetics and Genomes vol 5no 1 pp 235ndash245 2009

[22] I Lavrentieva N E Broude Y Lebedev et al ldquoHigh poly-morphism level of genomic sequences flanking insertion sitesof human endogenous retroviral long terminal repeatsrdquo FEBSLetters vol 443 no 3 pp 341ndash347 1999

[23] Y-G Liu and Y Chen ldquoHigh-efficiency thermal asymmet-ric interlaced PCR for amplification of unknown flankingsequencesrdquo BioTechniques vol 43 no 5 pp 649ndash656 2007

[24] I-T Hwang Y-J Kim S-H Kim C-I Kwak Y-Y Gu andJ-Y Chun ldquoAnnealing control primer system for improvingspecificity of PCR amplificationrdquo BioTechniques vol 35 no 6pp 1180ndash1184 2003

[25] P D Siebert A Chenchik D E Kellogg K A Lukyanov andS A Lukyanov ldquoAn improved PCR method for walking inuncloned genomic DNArdquo Nucleic Acids Research vol 23 no 6pp 1087ndash1088 1995

[26] D-L Guo X-G Hou and J Zhang ldquoSequence-related ampli-fied polymorphism analysis of tree peony (Paeonia suffruticosaAndrews) cultivars with different flower coloursrdquoThe Journal ofHorticultural Science and Biotechnology vol 84 no 2 pp 131ndash136 2009

[27] P Vos R Hogers M Bleeker et al ldquoAFLP a new technique forDNA fingerprintingrdquo Nucleic Acids Research vol 23 no 21 pp4407ndash4414 1995

[28] B J Bassam G Caetano-Anolles and P M Gresshoff ldquoFastand sensitive silver staining of DNA in polyacrylamide gelsrdquoAnalytical Biochemistry vol 196 no 1 pp 80ndash83 1991

[29] M McPherson and S Moslashller ldquoOptimization of PCRrdquo in PCRM McPherson and S Moslashller Eds pp 67ndash87 BIOS ScientificPublishers Oxfordshire UK 2000

[30] G Abrusan N Grundmann L Demester and W MakalowskildquoTEclassmdasha tool for automated classification of unknowneukaryotic transposable elementsrdquo Bioinformatics vol 25 no10 pp 1329ndash1330 2009

[31] S Priant T Heyman M L Wilhelm and F X WilhelmldquoExtended interactions between the primer tRNAiMet andgenomic RNA of the yeast Ty1 retrotransposonrdquo Nucleic AcidsResearch vol 24 no 3 pp 441ndash449 1996

[32] A Suoniemi K Anamthawat-Jonsson T Arna and A HSchulman ldquoRetrotransposon BARE-1 is a major dispersedcomponent of the barley (Hordeum vulgare L) genomerdquo PlantMolecular Biology vol 30 no 6 pp 1321ndash1329 1996

[33] S Steinbiss U Willhoeft G Gremme and S Kurtz ldquoFine-grained annotation and classification of de novo predicted LTRretrotransposonsrdquoNucleic Acids Research vol 37 no 21 ArticleID gkp759 pp 7002ndash7013 2009

[34] P Poczai I Varga M Laos et al ldquoAdvances in plant gene-targeted and functional markers a reviewrdquo Plant Methods vol9 no 1 6 pages 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


transposition [2] PBS is adjacent to the 51015840-LTR and used toprime the reverse transcriptase-catalyzed synthesis of minus-strand cDNA [10] PPT is located upstream of the 31015840-LTR andis a site of plus-strand synthesis initiation [2 16] Anotherimportant element of RTNs RNase H is responsible forthe degradation of the RNA template in the DNA-RNAhybrid The specific structural features of LTR-RTNs havebeen described by Wicker et al [17]

Pearce et al [14] firstly reported a novel technique forrapid isolation of plant Ty1-copia terminal repeat sequencesof RTNs based on the consensus amino acids of RNase Hmotif After that various isolation methods of LTR-RTNshave been developed based on the conserved primers [10 14]and genome walking methods [15 18ndash21] These methodshave different principles and efficiencies but some of themeither need probe hybridization or need enzyme digestionand adapter ligationThese lead to the tedious procedures andinfluence the isolation efficiency Compared with the existingmethods for isolation of LTR sequence the key factor is theefficiency of chromosome walking which is used to cloneunknown LTR region based on the conserved RNase H orPPT motif of RTNs [15 18ndash22]

In this study a new easy fast and efficient isolationmethod of RTN-LTRs was proposed and validated in treepeonyThis method combined the characteristics and advan-tages of high-efficiency thermal asymmetric interlaced PCR(hiTAIL-PCR) [23] annealing control primer (ACP) system[24] and suppression PCR [22 25] method It is simple low-cost and highly efficient which is just conducted throughthree rounds of PCR and does not need any restrictionenzymes and adapters much less the hybridizations

2 Materials and Methods

21 Materials and DNA Extraction Tree peony (Paeonia suf-fruticosa Andrews) cultivar ldquoLuoyanghongrdquo was used toisolate the LTR sequenceThe fresh leaveswere collected fromthe Luoyang National GenBank of Tree Peony China TotalDNAwas extracted from the fully expanded true leaves usingthe CTAB method [26]The material information used forSSAP analysis is shown in Table 1

22 PCR Amplification The components of PCR reactionand PCR program of three rounds were presented in Tables2 and 3 respectively The nested RNase H primers used inthis study were according to that of Pearce et al [14] TheACP primers were followed as that of Hwang et al [24] TheUP (universal primer) primer was designed based on theprimers of suppression PCR [25] PPT primer was designedto be degenerate primer based on the sequence characteristicsof PPT of RTNs [16] The corresponding primers sequencesare shown in Table 4 PCR products from the third roundof PCR were cloned into the PMD-18T vector using the TACloning Kit (TaKaRa Dalian China) The ligation productswere transformed into DH5120572 competent cell and positiveclones were sequenced by Sun Biotech Co (Beijing China)

23 Sequence Analysis The nature of cloned sequences wasconfirmed by performing similarity searches with knownRTNs sequences from other plants in NCBI database usingBLASTN BLASTX and TBLASTX algorithms with thedefault parameters GenomicDNA sequences were depositedin the GenBank databases Multiple DNA sequence align-ments were carried out using Lasergene 80 with MegAlignmodule by Jotun Hein method The LTR and PPT segmentsof the sequence were identified by comparing with the knownstructural characteristics of LTR and PPT regions of RTNs[14 15]

24 SSAP Analysis SSAP amplification was performed asdescribed by Bousios et al [19] SSAP analysis was alsocarried out twice for each primer pair in order to check theconsistency and reproducibility of the SSAP markers Thesequences of SSAP adapters and adapter primers were thesame as Vos et al [27] RTN primers were designed basedon the isolated LTR sequences according to the method ofWaugh et al [8] Selective amplification was conducted witha RTNs primer in combination with eitherMse I + 3 or EcoRI + 3 [27] The resulting bands were detected using the silverstaining protocol following the method of Bassam et al [28]

3 Results and Discussion

31 The Primer Design Principle of the NewMethod The suc-cess of PCR amplification relies on the binding specificitythat is a primer anneals to its target sequences There-fore it is important to optimize this molecular interaction[29] In order to improve the isolation efficiency of LTRsequence the method of high-efficiency TAIL-PCR (hiTAIL-PCR) presented by Liu and Chen [23] was firstly consideredwhich allows efficient amplification of large unknown targetsequences [23] The longer AD (LAD) primers of hiTAIL-PCR are 33 or 34 nucleotides which contained six or sevendegenerate nucleotides and four fixed nucleotides at the31015840 ends The arbitrary 31015840 four-base sites have a moderatefrequency (on average 256 bp at a time) to anchor the LADsto the unknown target regions Three nested specific primersets based on the known sequences were used to conductgenome walking in combination with the LAD primers Thespecific PCR products were amplified through the thermalasymmetric interlaced PCR programs and subsequent PCRreactions In order to employ hiTAIL-PCR in the newmethod the nested specific primers in the original hiTAIL-PCR were replaced by two nested RNase H primers [14] andone PPT degenerate primer based on the conserved motifof RNase H and PPT of RTNs At the same time it is a keyfactor for the success of PCR amplification that the primerscould bind to its target sequences or not at the period ofannealing Therefore the optimization of primer structureis very crucial for PCR amplification The primer annealingtemperature determines that the primer completely bindsto the template or partially mismatches the template in oneor several bases Consequently the adjustment of primerannealing temperature could improve the combination speci-ficity of primers and template Hwang et al [24] designed









the first PCR



















First PCR round





Primers Sequence


ACP primer









LTRIR IR

Gag

3998400

3998400

5998400

5998400

5998400

3998400

3998400

5998400

INT RTGenomic DNA

First amplification

RNase H1 primer


Target products

Product A

+

+


Nested primer PPT

Universal primer UP

Universal primer UP


No products

Formation

Stem-loop


RNase H

RNase H

RNase H

RNase H

RNase H1

RNase H2

PPT

PPT PPT

PPT

LTR

RNase H PPT LTR


Product B

Product C

PPT

PPT

PPT


Third amplification

LTR

LTR LTR

LTR

LTR

LTR

ACP primer

PR

3998400

3998400

5998400

5998400

3998400

5998400


2000 bp

500bp

100 bp









KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H


5998400 3

998400














500bp

140bp

20bp













4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









the first PCR



















First PCR round





Primers Sequence


ACP primer









LTRIR IR

Gag

3998400

3998400

5998400

5998400

5998400

3998400

3998400

5998400

INT RTGenomic DNA

First amplification

RNase H1 primer


Target products

Product A

+

+


Nested primer PPT

Universal primer UP

Universal primer UP


No products

Formation

Stem-loop


RNase H

RNase H

RNase H

RNase H

RNase H1

RNase H2

PPT

PPT PPT

PPT

LTR

RNase H PPT LTR


Product B

Product C

PPT

PPT

PPT


Third amplification

LTR

LTR LTR

LTR

LTR

LTR

ACP primer

PR

3998400

3998400

5998400

5998400

3998400

5998400


2000 bp

500bp

100 bp









KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H


5998400 3

998400














500bp

140bp

20bp













4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




First PCR round





Primers Sequence


ACP primer









LTRIR IR

Gag

3998400

3998400

5998400

5998400

5998400

3998400

3998400

5998400

INT RTGenomic DNA

First amplification

RNase H1 primer


Target products

Product A

+

+


Nested primer PPT

Universal primer UP

Universal primer UP


No products

Formation

Stem-loop


RNase H

RNase H

RNase H

RNase H

RNase H1

RNase H2

PPT

PPT PPT

PPT

LTR

RNase H PPT LTR


Product B

Product C

PPT

PPT

PPT


Third amplification

LTR

LTR LTR

LTR

LTR

LTR

ACP primer

PR

3998400

3998400

5998400

5998400

3998400

5998400


2000 bp

500bp

100 bp









KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H


5998400 3

998400














500bp

140bp

20bp













4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


LTRIR IR

Gag

3998400

3998400

5998400

5998400

5998400

3998400

3998400

5998400

INT RTGenomic DNA

First amplification

RNase H1 primer


Target products

Product A

+

+


Nested primer PPT

Universal primer UP

Universal primer UP


No products

Formation

Stem-loop


RNase H

RNase H

RNase H

RNase H

RNase H1

RNase H2

PPT

PPT PPT

PPT

LTR

RNase H PPT LTR


Product B

Product C

PPT

PPT

PPT


Third amplification

LTR

LTR LTR

LTR

LTR

LTR

ACP primer

PR

3998400

3998400

5998400

5998400

3998400

5998400


2000 bp

500bp

100 bp









KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H


5998400 3

998400














500bp

140bp

20bp













4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


KC519445

PBSLTR

ORFPol

Gag PR

PPT

PPT

LTR

LTR

UP

INT RT RNase H


5998400 3

998400














500bp

140bp

20bp













4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Conclusion




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article A Simple and Efficient Method to Isolate ...

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Transcript of Research Article A Simple and Efficient Method to Isolate ...