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

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 was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.).

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an 'orphan crop legume'. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation's Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an 'orphan legume crop' to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.