Illumina Beadxpress-based SNP assays for Germplasm Diversity Studies in Chickpea and Pigeonpea

Chickpea and pigeonpea are leading legumes that contribute more than 20% protein to human diet. Single nucleotide polymorphisms (SNPs) have become an ideal marker system due to their greater abundance in the plant genomes. In cases like marker-assisted selection (MAS) where only few markers are required for genotyping a set of potential lines, a cost-effective SNP genotyping platform is required. In this scenario GoldenGate assays based on VeraCode technology using Illumina BeadXpress platform seems to be a cost-effective. In the case of chickpea and pigeonpea, >10,000 SNPs have been identified using next-generation sequencing (NGS) technologies in earlier studies. Based on high polymorphism information content (PIC) and assay designing tool (ADT) scores, two sets of SNPs one each for chickpea (96 SNPs) and pigeonpea (48 SNPs) were designed for custom Oligo Pool Assays (OPAs) for Illumina BeadXpress genotyping platform. These OPAs were used to genotype reference sets (ca. 300 genotypes each) of both the legumes. All SNPs were successfully scored in the reference set, with less than 1% missing data (chickpea) and around 1-2% missing data in pigeonpea. The mean PIC value of SNP markers was 0.31 and 0.32 in chickpea and pigeonpea respectively. No unique pattern of grouping in chickpea reference set was observed based on neighbour joining tree constructed, while two major groups are observed in case of pigeonpea reference set. Our results demonstrated the usefulness of SNP assays in diversity analysis. Further, the polymorphic markers identified in the present study can be integrated on to respective genetic maps.