Development of novel SNP/InDel markers through amplicon sequencing in dolichos bean (Lablab purpureus L.)

Abstract:

Despite the advances made through genomics research in Dolichos bean, the availability of informative molecular tools is still limited. The expressed sequence tags represent the robust sequence resource for the development of novel genic markers that can be exploited for gene discovery, genome annotation and comparative genomics. Here we first time report the development of novel EST-derived SNP and InDel markers through amplicon sequencing of cross-transferable cowpea EST-SSR primers in Lablab bean. Total of four polymorphic cowpea EST-SSRs viz., CP2, CP8, CP29, and CP435, with a high transferability rate in Dolichos bean were identified. Sequenced total 18 genic fragments from nine parental genotypes, which resulted in the identification of 59 SNPs and 16 InDels. Based on these sequence variations, we successfully designed and validated eight allele-specific markers i.e. Met 2, AF 287258-1, AF 285278-2, AF 151961, D 13557, CP 5, CP16 and CP 43, which are targeting SNPs/InDels in Lablab bean. Further, to prove their immediate utility for trait mapping the parental polymorphic markers were genotyped on F2 segregating lines derived from the two crosses i.e. HA4 × CPI31113 and HA4 × CPI60216. Segregation analysis revealed that these markers exhibited a typical Mendelian segregation ratio of 1:2:1 with co-dominant allelic patterns for SNP/InDels. This confirmed the immediate utility of such novel genic markers for future genomics-assisted breeding applications in Dolichos bean.