http://oar.icrisat.org/id/eprint/12 2013-09-16T09:35:59Z info:eu-repo/semantics/descriptiveMetadata http://oar.icrisat.org/12/ Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh] Dutta, S Kumawat, G Singh, B P Gupta, D K Singh, Sangeeta Dogra, V Gaikwad, K Sharma, T R Raje, R S Bandhopadhya, T K Datta, S Singh, M N Bashasab, F Kulwal, P Wanjari, K B Varshney, R K Cook, D R Singh, N K Pigeonpea Background: Pigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping. Results: In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population. Conclusion: We developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers BioMed Central 2011 Article PeerReviewed application/pdf en http://oar.icrisat.org/12/1/varsh-2011-aupay.pdf Dutta, S and Kumawat, G and Singh, B P and Gupta, D K and Singh, Sangeeta and Dogra, V and Gaikwad, K and Sharma, T R and Raje, R S and Bandhopadhya, T K and Datta, S and Singh, M N and Bashasab, F and Kulwal, P and Wanjari, K B and Varshney, R K and Cook, D R and Singh, N K (2011) Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]. BMC Plant Biology, 11 (1). pp. 17-29. ISSN 14712229 http://dx.doi.org/10.1186/1471-2229-11-17 info:eu-repo/semantics/objectFile info:eu-repo/semantics/humanStartPage