ISSN 0096�3925, Moscow University Biological Sciences Bulletin, 2012, Vol. 67, No. 2, pp. 48–51. © Allerton Press, Inc., 2012.Original Russian Text © Z.G. Kokaeva, A.V. Aleshin, Y.I. Berezov, 2012, published in Vestnik Moskovskogo Universiteta. Biologiya, 2012, No. 2, pp. 9–12.

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INTRODUCTION

The structure and polymorphism of peas' mobilegenetic elements, first of all of retrotransposons, hasbeen actively studied recently. Many molecular mark�ers have been obtained on the basis of retrotransposons[1–3]. The information on retrotransposon polymor�phism allows for a conclusion on the origin of differentsubspecies and sorts [4–6]. The markers based on ret�rotransposons are very convenient for genetic map�ping and study of genetic diversity of plants [2].

Due to the usage of common markers, we managedto build a “consensus” map of chromosomes of peas[7] that united morphological and molecular loci.However, a detailed chromosome map of the pea hasnot yet been made, and additional search for new mor�phological and molecular markers and their localiza�tion is required.

The process of development of new marker systemsthat consists in detection of presence or absence of ret�rotransposon insertions occurs constantly. Recently,four methods are used most often: SSAP (sequenceSpecific Amplication Polymorphism) [8], IRAP(Inter Retrotransposon Amplified Polymorphism) [3],REMAP (retrotransposon�Microsatellite AmplifiedPolymorphism) [3], RBIP (Retrotransposon BasedInsertion Polymorphism) [1]. RBIP (the methodwhich reveals polymorphism of certain insertions ofretrotransposon in the genome) differs from all of theabove�mentioned methods in that it allows studyingcodominant allelic variants as well as using significantamount of the sample for the dot�blot analysis. At theDepartment of Genetics (Biological Faculty, MoscowState University) DNA�marker kits were produced

using IRAP and REMAP methods for reliable identi�fication of sorts and lines of pea [9, 20]. Systematicwork is being conducted on mapping of the peagenome, for which RAPD and ISSR�markers werepreviously effectively used: the study resulted in fullmaps of all coupling groups of pea which were built ontwo mapping populations (Chil 15 × WL1238 andP64 × WL1238) that contain more than 200 molecularmarkers [11].

PDR1 was the first group of Ty1�copia retrotrans�posons that was studied in the genome of pea [12]where about 200 of its copies were revealed. It refers tothe group of small retrotransposons (3925 bp) withtypical gene order gag�pr�int�rt�maseH, and LTR(long terminal repeats) contain 156 bp.

The aim of the work is to study RBIP�insertionpolymorphism in the sorts, lines, and mutants of peausing seven pairs of specific RBIP�primers chosen forretrotransposon PDR1 and localization of revealedDNA�markers on genetic map of pea during geneticanalysis of F2 from the cross (Chil 15 × WL1238).

MATERIALS AND METHODS

In the work, we used 50 samples of planting peathat belong to sorts and lines of different areas of selec�tion as well as a population of F2 hybrids (Chil 15 ×WL1238), which includes 110 plants. Parental lines:Chil15—chlorophyll mutant that was obtained bytreatment of the seeds of sort Torsda with ethyl�methansulphate and characterized by greenish colorof leaves; marker line WL1238, which has greatamount of molecular markers.

Analysis of PDR1 Retrotransposon Insertionsin the Pea (Pisum Sativum L.)

Z. G. Kokaeva, A. V. Aleshin, and Y. I. BerezovDepartment of Biology, Moscow State University, Moscow, Russiae�mail: zaremak@inbox.ru, starshina_pp@mail.ru, phlora@list.ru

Received December 23, 2010

Abstract—We research polymorphism of PDR1 insertion grades, lines, and mutants of peas by means ofseven pairs of specific RBIP�primers. Seven PDR1 insertions loci are identified in the studied forms of peas.We establish the high level of polymorphism of PDR1 retrotransposon inserts with primers RBIP2,RBIP1794, and RBIP64, that has made 92, 82.98 and 78%, respectively. The genetic analysis in a populationof F2 hybrids (Chil 15 × WL1238) is conducted. RBIP700 polymorphic fragment is localized on a geneticmap of peas in the third group of coupling.

Keywords: retrotransposon, molecular marker, genetic mapping, SSAP, IRAP, REMAP, RBIP

DOI: 10.3103/S0096392512020071

MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 67 No. 2 2012

ANALYSIS OF PDR1 RETROTRANSPOSON INSERTIONS IN THE PEA 49

The procedure of extraction of plant DNA wasconducted by method [13] with slight modifications.Reaction mixture of volume 25 µl contained 5 µl ofDNA solution, 2.5 U Taq�polymerase (Sileks M.),250 µl of each dNTPs (Sileks M.), 0.5 µM of eachprimer, and 2.5 µl of 10x standard PCR�bufer(Sileks M.). Amplification was conducted in a Tercikamplificator made by DNK�Tekhnologii using the fol�lowing program: 1. Preliminary denaturation 94°C,1 min 30 s; 5 cycles (94°C, 1 min; 54°C, 40 s; 72°C,1 min); 30 cycles (93°C, 1 min; 54°C, 40 s; 72°C1 min), final elongation 72°C, 10 min. The primerswere synthesized in Sintol. The temperature of primerannealing was assessed using computer programOLIGO. The separation of DNA fragments obtainedas a result of amplification was conducted by standardelectrophoresis in 2% agarose gel (Amresco) in TBE�buffer with the following staining with ethidium bro�mide (final concentration 0.001%). Intensity of elec�tric field during electrophoresis was 3 B/sm. Afterelectrophoresis, the gel was analyzed under UV lightand captured using a digital camera.

For the genetic analysis of splitting in F2, w usedMapmaker 3.0 software developed in the WhiteheadInstitute (United States) that is widely used in the con�temporary works on mapping.

RESULTS AND DISCUSSION

In this work, we conducted a study of distributionof RBIP insertions in sorts, lines, and mutants of peausing seven pairs of specific RBIP�primers chosen forPDR1 retrotransposon [1]. The primers are character�ized in Table 1.

As a result of this work, seven loci of RBIP�inser�tions in the studied forms of pea were identified.Figure 1 presents the result of electrophoretic separa�tion of amplified products of PCR of one locus RBIPABC in sorts, lines, and mutants of pea. Amplificationproduct 537 bp in size corresponds to the presence ofretrotransposon in this locus and that of 243 bp corre�sponds to the absence of the retrotransposon. Thus,both alleles of this locus are amplified. The results

Table 1. RBIP primers

Primer name Equence Vmixture forthe reaction, µl

Birte�x16�F 54 CTTACCACCAAGCGCGCGAC 0.57

Birte�x16�R 54 AGGCTTCTGATCCAACCAG 0.39

Birte�X5�F 54 CGAACTCATATAGTAATACTTAG 0.43

Birte�X5�R 54 CTCTTATGTCTTGCTGAGGGTG 0.60

MKRBIP2�F 54 TGTAGATGGTATGCTGTACTC 0.42

MKRBIP2�R 54 ATCTACCATTTATTTCCACGC 0.54

MKRBIP4�F 54 TTGATACTaTCATGCTTGGCC 0.50

MKRBIP4�R 54 ATGAGAGTTGAGTTATATTCC 0.40

1794�2�F 54 GGGCCATGTACGACACATTC 0.33

1794�2�R 54 GAGGAAATAAGAATGGTAGAGCATC 0.52

64�x45�F 54 CAAAGTATAACGTGTATCAAG 0.39

64�x45�R 54 GTCATCGTCCAAACACACTC 0.38

RBIP A 65.8 CCCATTGATTCTCGTCTCAAGAC 0.14

RBIP B 65.3 GGCTTTGACTAATGGACCTC 0.16

RBIP C 64.5 TCCTGTTCAGCATGACAGGAC 0.11

tannealing0

537

243

M1 2 3 5 64 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 34 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 M

Fig. 1. RBIP�spectra of sorts, lines, and mutants of pea obtained with RBIP ABC primer. 1–49 are individual plants of the studiedform of pea (mutant Khl.�13 is absent). M�DNA ladder 100 bp + 1.5 kb. The arrows indicate alleles of RBIP ABC locus alleles.

50

MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 67 No. 2 2012

KOKAEVA et al.

obtained on all studied RBIP�loci are presented inTable 2.

The high level of polymorphism of PDR1 ret�rotransposon with primers RBIP2, RBIP1794, andRBIP64, which made 92, 82.98, and 78%, respec�tively, was established. These data well correlate to theresults obtained in the works [4, 6].

With the aim to localize DNA�markers with poly�morphic sequences on the coupling groups, a map ofpea genetic analysis in F2 hybrid populations from thecross (Chil15 × WL1238) was build. In this popula�tion, an analysis with a larger number of randommarkers was conducted, and saturated maps of peacoupling groups were built [11, 15].

During our work, we managed to localize on themap polymorphic fragment RBIP4700, which wasobtained during the analysis of insertions of PDR1 ret�rotransposon and that differs between parental linesChil15 and Wl1238 (Fig. 2). After checking the reli�ability of hypotheses on monogenic study of polymor�phic variants, the data on splitting were introduced incommon primary files in Mapmaker format for thementioned crosses, which contain previously obtaineddata on plant phenotype on qualitative morphologicalfeatures and a number of random markers (125 mark�ers in each mapping population) [11]. After that, tak�ing into account the information on chromosomallocalization of genes of morphological features [7] and

those genes that were mapped before [15, 16], we con�ducted a search for the cases of coupling of this locusRBIP4700 with the other genes (CAPS markers) andrandom markers segregating in mapping populations.

Using the MAPMAKER 3.0 software we calcu�lated genetic distances between molecular markers

Table 2. Molecular genetic analysis of PDR1 retrotransposon insertions of pea using the RBIP method

Method PrimersNumber of retrotrans�

posons in the locus (number of forms)

Absence of retrotrans�poson in the locus (number of forms)

Polymorphismgrade, %

RBIP 1794 8 39 82.98RBIP Birte 16 46 4 8RBIP Birte X5F 23 26 53.06RBIP RBIP 2FR 4 46 92RBIP RBIP 4FR 36 11 23.4RBIP RBIP 64FR 11 39 78RBIP RBIP ABC 38 11 22.45

700

400

1 2 M

Fig. 2. Electrophoregramm of the results of RBIP�analy�sis. 1—line WL1238; 2—line CHil15. M�DNA ladder100 bp + 1.5 kb. The arrows indicate RBIP4 locus alleles.

PhPypC

Rnp33

6.8

9.4

Rb4

Sodmt

Adh1

12.7

10.7

Le

PK4

CipPor

PepsEdl1

B

CbI

PhlC

Rnp33

Adh1

Sodmt

Chi115

GprAII

TubA1

GprBRb

0.8(27.87)

8.7(9.97)

17.6(5.55)

11.1(4.26)

12.7(7.19)

8.7(8.7)

15.6(5.49)

7.7(9.55)

23.9(4.26)

11.6(1.15)

6.6(6.02)3.4

(20.91)6.0

(16.17)

Fig. 3. Fragment of map of III GS with RBIP4 markerslocalized on it (Rb4) (on the left) and comparison of thisfragment with GS map III built on population Chil15 ×WL1238 (to the right) [15].

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ANALYSIS OF PDR1 RETROTRANSPOSON INSERTIONS IN THE PEA 51

and genes with which they are coupled and identifiedmutual localization of the markers. As a result of theconducted work on the basis of population Chil15 ×WL1238, RBIP4700 fragment was localized in groupIII of the coupling of pea. The locus is located in theupper arm of the chromosome at 12.7 cM from themarker Adh1, 9.4 cM from the marker Rnp33, and34.1 cM from gene Chil15 (Fig. 3).

CONCLUSIONS

The study of polymorphism of PDR1 retrotrans�posons insertions in sorts, lines, and mutants of thepea was conducted and seven loci of PDR1 insertionsin the studied forms of peas were identified. The highlevel of polymorphism of PDR1 retrotransposoninsertions with the primers RBIP2, RBIP1794, andRBIP64, which made 92, 82.98, and 78%, respec�tively, was established. Molecular marker RBIP4700was localized in the third group of the coupling of pea.The locus is located in the upper arm of the chromo�some between the markers Adh1 and Rnp33 and at34.1 cM from gene Chil15, which conditions chloro�phyll deficiency. Thus, the obtained marker RBIP4700may be used as an anchor marker in the next studies.

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