The 96 plex and 48plex SNPs assays could successfully characterize reference sets of chickpea and pigeonpea respectively. These SNPs can easily be employed in molecular breeding programmes where only few markers are required for genotyping of selected set of genotypes. BeadXpress Reader at Genotyping Services Laboratory, ICRISAT Roorkiwal M 1 , Sawargaonkar SL 1 , Thudi M 1 , Saxena RK 1 , Upadhyaya HD 1 , Varshney RK 1,2,* 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 genotyping method. 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 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 in case of chickpea and around 1-2% missing data in pigeonpea. The mean polymorphism information content (PIC) of SNP markers in case of chickpea was 0.31 and 0.32 in pigeonpea. No unique pattern of grouping in chickpea reference set was observed based on neighbour joining tree constructed. However, in the case of pigeonpea two major groups are observed with SNPs data. Our results indicate that Illumina BeadXpress based system provides a cost effective SNP genotyping method for selected set of potential SNP markers and can easily be used for diversity studies. Financial support from Department of Biotechnology, Government of India and CGIAR Generation Challenge Programme (GCP) is gratefully acknowledged. Abstract Acknowledgements Summary Highly polymorphic SNPs Utility of designed SNP plex in breeding applications Mapping population Population Traits No. of SNPs Chickpea (96-plex) ICC 283 × ICC 8261 RIL Root traits 57 ICC 3137 × ICC 72933 RIL Helicoverpa resistance 57 ICC 4958 × ICC 1882 RIL Root traits 56 DCP 92-3 × ICC 4958 MABC Drought tolerance 48 ICC 6362 × ICC 1431 RIL Salinity tolerance 44 Pigeonpea (48-plex) ICPL 20096 × ICPL 332 RIL Fusarium wilt 28 ICP 8863 × ICC 20097 RIL Sterility mosaic disease 14 Designed 96 and 48 OPAs for chickpea and pigeonpea respectively are being used for diversity analysis, trait mapping and marker-assisted selection SNP genotyping using Illumina BeadXpress for germplasm diversity studies in chickpea and pigeonpea 0 5 10 15 20 25 30 35 40 45 No. of SNPs PIC value range 0 5 10 15 20 25 30 35 40 45 No. of SNPs PIC value range Chickpea-96 SNPs Pigeonpea-48 SNPs SNP SSR SNP SSR Genetic structure analysis of chickpea reference set Genetic structure analysis of pigeonpea reference set Allele calling using GenomeStudio Data Analysis Software SNP genotyping using BeadXpress Reader • 96-plex (chickpea) and 48-plex (pigeonpea) OPAs for SNP assays was designed based on polymorphism among the cultivated chickpea and pigeonpea genotypes and high ADT scores • 288 and 256 genotypes of chickpea and pigeonpea reference set respectively were genotyped using the OPAs • All 96 and 48 SNPs gave clear genotyping and are highly polymorphic across their respective reference set • The success rate of genotyping is high 98% in chickpea and 90% in pigeonpea • SNPs in both species could effectively characterize the reference sets compared to SSR markers 1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India; 2 Generation Challenge Programme (GCP), c/o CIMMYT, Mexico DF, Mexico; *Address for correspondence: [email protected] B: Breeding line L: Landrace W: Wild relative