Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]

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

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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

Item Type: Article
Agro Tags: <b>Agrotags</b> - genetics | locus | polymorphism | genomes | genes | dna | planting | biodiversity | grain legumes | genotypes <br><b>Fishtags</b> - NOT-AVAILABLE<br><b>Geopoliticaltags</b> - india | delhi | usa | maine | centre
Subjects: Mandate crops > Pigeonpea
Depositing User: Siva Shankar
Date Deposited: 17 May 2011 07:57
Last Modified: 16 Sep 2013 09:35
Official URL:
Funders: Indian Council of Agricultural Research, National Science Foundation (NSF), USA, Government of India - Department of Biotechnology
Acknowledgement: This study was financially supported by the Pigeonpea Genomics Initiative (PGI) of the Indian Council of Agricultural Research (ICAR), New Delhi under the framework of Indo-US Agricultural Knowledge Initiative (AKI). Contribution of Doug Cook was supported by the National Science Foundation (NSF), USA. GK acknowledges fellowship support from Department of Biotechnology, Government of India.
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