To increase the number of polymorphic simple sequence repeat markers (SSRs) in chickpea, a genomic library was constructed, and the SSRs derived from this approach are characterized. A genomic DNA library from the chickpea genotype ICC 4958 was constructed after digesting total DNA of ICC 4958 with MBO/Sau and TaqI at University of Frankfurt, Germany. The study increases the existing SSR repertoire in chickpea, which will help to enhance the coverage of linkage maps especially in intraspecific crosses where marker polymorphism is found to be very less

Hoisington, D.A.

Horres, R.

Jayashree, B.

Jungmann, R.

Kahl, G.

Molina, C.M.

Nayak, S.N.

Swamy, P.

Varshney, R.K.

Winter, P.

English

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An Open Access Journal published by ICRISATSAT eJournal ⏐ ejournal.icrisat.org - 1 - December 2007 ⏐ Volume 5 ⏐ Issue 1Extending the repertoire of microsatellite markers for genetic linkagemapping and germplasm screening in chickpeaRK Varshney1*, R Horres2, C Molina3, S Nayak1, R Jungmann3, P Swamy1, P Winter2, B Jayashree1, G Kahl3 andDA Hoisington11. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India2. GenXPro GmbH, Frankfurter Innovationszentrum Biotechnologie (FIZ), Altenhöferallee 3, D-60438 Frankfurt am Main, Germany3. University of Frankfurt, Max von Laue Str. 9, D-60439 Frankfurt am Main, Germany*Corresponding author: r.k.varshney@cgiar.orgMolecular markers and genetic linkage maps are theprerequisites for undertaking molecular breedingactivities. However, the progress towards developmentof a reasonable number of molecular markers has beenvery slow in cultivated species of chickpea (Cicerarietinum). One of the main reasons for this may beattributed to the low level of genetic diversity present inthe cultivated gene pools of these species, at least withthe detection tools that are currently available. Amongvarious molecular markers currently available,microsatellite or SSR (simple sequence repeat) markersare often chosen as the preferred markers for a variety ofapplications in breeding because of their multi-allelicnature, co-dominant inheritance, relative abundance andextensive genome coverage (Gupta and Varshney 2000).As a result, several hundred of SSR markers have beendeveloped in chickpea (Hüttel et al. 1999, Winter et al.1999, Sethy et al. 2003, 2006, Lichtenzveig et al. 2005,Choudhary et al. 2006). Despite the availability ofhundreds of SSR markers, the density of the intraspecificgenetic maps is still very low. With an objective toincrease the number of polymorphic SSR markers inchickpea, a genomic library was constructed and SSRsderived from this approach were characterized in thisstudy.A genomic DNA library from the chickpea genotypeICC 4958 was constructed after digesting total DNA ofICC 4958 with MBO/Sau and TaqI at University ofFrankfurt, Germany. Selected fragments of 800–1200 bpwere purified and ligated to Promega pGEM 3Z(f) vector(Promega, USA). Plasmids were recovered andelectroporated into Escherichia coli Sure strain (DH10B,Stratagene) at 1.8 kV/cm using a BIORAD Gene pulserand cloned in DH10B (E. coli). After plating about400,000 clones at a density of 20,000 colonies per plateon 24 cm × 24 cm petri dishes (Nunc, Germany),masterplates [384-well micro-titre plates (MTPs)] weregenerated. Replicas were taken from these master platesand spotted in duplicate on (positively charged) PVDF-Macroarrays at RZPD GmbH, Berlin, Germany (http://www.rzpd.de). Subsequently, the hybridization ofmacroarrays with the 3’end-labeled synthetic repeats(GA)10 and (TAA)10 and screening of positive cloneswere performed at GenXPro GmbH. Clones with positivesignals were grown on LB (Luria Bertani) agar plateswith ampicillin overnight at 37°C. Aliquots of thesecolonies were used for colony PCR (polymerase chainreaction). Subsequently, a total of 359 clones with strongto moderate signals for the two SSRs were selected.The bacterial colonies were grown overnight at 37°Cin 5 ml of Luria Broth medium with 100 μg ml-1 ampicillinin an orbital shaker (Thermo Electron Corporation, USA)at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India. The plasmidDNA from individual clones was isolated using standardalkaline lysis method (Sambrook and Russell 2001).After checking the quality of the plasmid DNA on 0.8%agarose gel, the clones were sequenced at ICRISAT andMacrogen (South Korea) using the BigDye Terminatorcycle sequencing kit on an ABI3700/ABI3730XLTable 1. Summary on SSR search in the microsatelliteenriched genomic library.Total number of sequences examined 457Total size of examined sequences (bp) 286,718Total number of identified SSRs 643Number of SSR containing sequences 299Number of sequences containing more than one SSR 165Number of SSRs present in compound form 221Citation : Varshney RK, Horres R, Molina C, Nayak S, Jungmann R, Swamy P, Winter P, Jayashree B, Kahl G andHoisington DA, (2007) Extending the repertoire of microsatellite markers for genetic linkage mapping and germplasmscreening in chickpea. Journal of SAT Agricultural Resarch 5(1).An Open Access Journal published by ICRISATSAT eJournal ⏐ ejournal.icrisat.org - 2 - December 2007 ⏐ Volume 5 ⏐ Issue 1(Applied Biosystems, USA). A total of 288 clones weresequenced at Macrogen in both directions using standardT7 promoter and SP6 primers and 576 sequences weregenerated. Nineteen clones were sequenced at ICRISATin only one direction by using M13-forward sequencingprimer. In total, 595 forward and/or reverse sequenceswere generated. Subsequently, all 595 raw sequenceswere trimmed using Sequencher program to remove theresidues of the vector sequences following standardparameters. The cleaned sequences were checked for thequality of run, by inspecting the chromatograms of thesequence data. In 23 cases the chromatogram showed badsequencing quality; hence those 23 sequence data wereremoved from the list. Further, the sequence residues lessthan 100 bp and more than 700 bp were removed in orderto ensure high quality sequence data. Sequences obtainedusing the T7 promoter and SP6 primers were subjected toContig Assembly Program (CAP3) to obtain the contigsequence (http://bioweb.pasteur.fr/seqanal/interfaces/cap3.html). Out of a total of 572 cleaned sequences (fromforward as well as reverse), only 115 contigs could begenerated, while 342 sequences remained singletons.Ultimately, a total of 457 sequences representing 115contigs and 342 singletons were screened for thepresence of SSR using a Perl5 script called MISA(MIcroSAtellite), available at http://pgrc.ipk-gatersleben.de/misa/. The MISA search tool is capable ofidentifying both perfect and compound SSRs.Mononucleotide (N) SSR with minimum 10 repeat unitsand di- (NN), tri- (NNN), tetra- (NNNN), penta-(NNNNN) and hexa- (NNNNNN) nucleotide with aminimum of four repeat units were considered. Thecompound microsatellites were considered with a minimumdistance of 100 bp. The sequence complementarity wasconsidered while classifying the microsatellites. Forinstance, the repeat motifs AG, GC, TC, CT were groupedin the same class. Following the above mentioned criteria,a total of 643 SSRs were identified in 299 sequences out of457 sequences representing 28.67 kb sequence data(Table 1). A total of 165 sequences contained more thanone SSR while 221 sequences were found to havecompound SSRs.In terms of the frequency of different SSRs identified,the di- and tri-nucleotide SSRs were the most abundant(39% and 40%, respectively). Mononucleotide SSRsoccurred in about 16% cases and tetranucleotide SSRs inabout 3%. Other types of SSRs had <1% representation(Fig. 1). In majority (467; 73%) of the 643 SSR motifs,the microsatellites were of less than 10 repeat unitsfollowed by those having repeat units between 10 and 20050100150200250<10 <20 <30 <40 >41Repeat unitsNumber of SSRs NNNNNNNNNNNNNNNNNNNNN050100150200250300N NN NNN NNNN NNNNN NNNNNNType of microsatelliteFrequency (%)Figure 2. Number of SSRs with different number of repeat units.Figure 1. Frequency of different SSR classes.An Open Access Journal published by ICRISATSAT eJournal ⏐ ejournal.icrisat.org - 3 - December 2007 ⏐ Volume 5 ⏐ Issue 1(137 cases; 21%). However, only very few microsatelliteswere observed in other classes of SSR, eg, 20-30, 30-40,etc. The distribution of different lengths of SSRs isshown in Figure 2.In order to develop SSR markers for chickpea genomeanalysis, all 299 sequences containing SSRs (including165 sequences where more than one SSR was present)were used for primer design using the PRIMER3program in a batch file. As a result, a total of 311 primerpairs were designed. For some sequences, primer pairswere designed for more than one SSR. Although thisincreases the redundancy, it ensures not to miss anypolymorphic SSR in a given sequence.At present, these SSR markers are being screened on apanel of 12 parental genotypes representing parentalgenotypes of six intraspecific (C. arietinum × C.arietinum) mapping populations – ICC 506EB × Vijay,ICC 3137 × IG 72953, ICC 3137 × IG 72933, ICC 283 ×ICC 8261, ICC 4958 × ICC 1882, ICCV 2 × JG 62 – andone interspecific reference mapping population C.arietinum ICC 4958 × C. reticulatum PI 489777.Markers showing polymorphism in above mentionedcombinations will be used to genotype the correspondingmapping populations to prepare linkage maps and traitmapping at ICRISAT. Another interspecific mappingpopulation C. arietinum ICC 6098 × C. reticulatumILWC 36 will be genotyped at Frankfurt. The developedresource of SSR markers is available upon request andhas been shared with several collaborators includingWashington State University, Pullman, USA (FredMuehlbauer), The Australian Centre for NecrotrophicFungal Pathogens, Murdoch University, Perth, Australia(Richard Oliver), the National Chemical Laboratory,Pune, India (Vidya Gupta) and the National ResearchCentre on Plant Biotechnology, New Delhi, India(Ramamurthy Srinivasan). In summary, this study increasesthe existing SSR repertoire in chickpea, which will help toenhance the coverage of linkage maps especially inintraspecific crosses where marker polymorphism isfound to be very less.Acknowledgment. We gratefully acknowledge the financialsupport received from Generation Challenge Programmethrough Tropical Legume I Project and Agri-SciencePark of ICRISAT under the umbrella of Israel-ICRISATcollaborative grant.ReferencesChoudhary S, Sethy NK, Shokeen B and Bhatia S.2006. Development of sequence-tagged microsatellitesite markers for chickpea (Cicer arietinum L.). MolecularEcology Notes 6:3–95.Gupta PK and Varshney RK. 2000. The developmentand use of microsatellite markers for genetic analysis andplant breeding with emphasis on bread wheat. Euphytica113:163–185.Hüttel B, Winter P, Weising K, Choumane W,Weigand F and Kahl G. 1999. Sequence-taggedmicrosatellite site markers for chickpea (Cicer arietinumL.). Genome 42:210–217.Lichtenzveig J, Scheuring C, Dodge J, Abbo S andZhang HB. 2005. Construction of BAC and BIBAClibraries and their applications for generation of SSRmarkers for genome analysis of chickpea (Cicer arietinum(L.)). Theoretical and Applied Genetics 110:492–510.Sambrook J and Russell DW. 2001. Molecular cloning.A laboratory manual. Edn 3. Cold Spring Harbor, NewYork, USA: Cold Spring Harbor Laboratory Press. 540 pp.Sethy NK, Choudhary S, Shokeen B and Bhatia S. 2006. Identification of microsatellite markers from Cicerreticulatum: molecular variation and phylogeneticanalysis. Theoretical and Applied Genetics 112:347– 357.Sethy NK, Shokeen B and Bhatia S. 2003. Isolation andcharacterization of sequence-tagged microsatellite sitesmarkers in chickpea (Cicer arietinum L.). MolecularEcology Notes 3:428–430.Winter P, Pfaff T, Udupa SM, Hüttel B, Sharma PC,Sahi S, Arreguin-Espinoza R, Weigand F,Muehlbauer FJ and Kahl G. 1999. Characterizationand mapping of sequence-tagged microsatellite sites inthe chickpea (Cicer arietinum L.) genome. Molecularand General Genetics 262:90–101.View publication stats

Extending the repertoire of microsatellite markers for genetic linkage mapping and germplasm

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Extending the repertoire of microsatellite markers for genetic linkage mapping and germplasm

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