Discovery of SNPs in important legumes through comparative genome analysis and conversion of SNPs into PCR-based markers.

Compared to cereal crops several legumes are less characterized at the genomic level and rightly referred as orphan crops. Transfer of knowledge between model and crop legumes allows development of orthologous pan-taxon genomic tools to benefit research on resource poor taxa. Here, we developed 278 intron flanking gene-specific markers by BLAST aligning pigeonpea (Cajanus cajan (L.) Millsp.) expressed sequence tags (ESTs) with complete genome sequence of barrel medic (Medicago truncatula). A random 192 PCR primer pairs representing loci across the haploid genome (n = 8) of barrel medic were tested on a few important members of legume family. The single copy amplification rates of 31.8% (peanut, Arachis hypogaea) to 77.6% (barrel medic) signifies the success of cross taxon primers and suggested their potential use in comparative legume genomics. Genetic diversity was assessed in 48 pigeonpea genotypes using 143 intron flanking markers which revealed 71 polymorphic markers with PIC values ranging from 0.04 to 0.45 with an average of 0.23 per marker. The PCR products of different varieties of pigeonpea, cowpea and chickpea were sequenced and aligned to find putative SNPs. Integration of these newly developed markers into genetic maps in resource poor legumes will not only aid in the map saturation but also in designing successful marker-assisted selection programmes.

Development and application of genomic resources for comparative and translational genomics in legumes through leveraging genomic sequence of Medicago truncatula.

The expressed sequence tags (ESTs) of common bean were BLAST aligned with barred medic genome sequence and developed 1196 conserved intron spanning primers (CISPs) to facilitate genetic studies in legumes. Randomly selected 288 CISPs, representing loci on barrel medic genome, were tested on 10 selected members of legume family. On the source taxa, the highest single copy amplification success rates of 61.8% (barrel medic) and 56.2% (common bean) was obtained. The success rate of markers was 54.5% in cowpea followed by 53.5% in pigeonpea and chickpea, signifying cross taxon amplification and their potential use in comparative genomics. However, relatively low percentages of primer set amplified (40-43%) in soybean, urdbean and peanut. Further, these primers were tested on different varieties of chickpea, pigeonpea and cowpea. The PCR products were sequenced and aligned which resulted in detection of 26 SNPs and eight INDeLs in cowpea, seven SNPs and two INDeLs in chickpea and 27 SNPs and 14 INDeLs in pigeonpea. These SNPs were successfully converted in to size variation for gel-based genotyping. The CISP markers developed in this study are expected to aid in map saturation of legumes and in marker-assisted selection for accelerated crop improvement.