Gene Discovery and Development of Functional Markers for MYMIV Resistance in Urdbean ( Vigna mungo L. Hepper)

ABSTRACT Mungbean Yellow Mosaic India Virus (MYMIV) is a major disease of urdbean (black gram) crop, which is one of India's most widely consumed pulses. In the present study, RNA‐Seq‐mediated differential gene expression analysis was conducted between a resistant variety (PU‐31) and a susceptible variety (LBG‐17) of urdbeans for the identification of resistant genes for the MYMIV disease. This resulted in the generation of a total of 827,157,878 clean reads through NovaSeq 6000 sequencing from the 12 samples of resistant and susceptible varieties evaluated under control (disease‐free) and treatment conditions (disease condition). A trinity‐based de novo assembly was developed with 553,889 coding regions of the urdbean genome. A set of 16 transcripts related to disease resistance that had high expression values in the present study was analyzed for differential gene expression using RT‐PCR. Among these, two validated transcripts, TRINITY_DN4785_c0_g1_i9 (F‐box/LRR resistance gene) and TRINITY_DN21430_c0_g3_i1 (Tobacco Mosaic Virus resistance gene), were used to develop DNA‐based PCR markers. Using Sanger sequencing, five SNPs were found for an “F‐box/LRR” resistance gene and a single SNP in the case of the “TMV” resistance gene. Using these SNPs, polymorphic PCR usable primers were designed and validated in a panel of urdbean genotypes. Utilizing SNP‐445 of the “TMV” resistance gene, CAPS (Cleaved Amplified Polymorphic Sequence) and TSP (Temperature Switch PCR) markers were developed, and a TSP marker using SNP‐361 was developed for the “F‐box/LRR” resistance gene. Several urdbean varieties examined in the present study were found to harbor resistance alleles of the “TMV” and “F‐box/LRR” genes, which likely contribute to the durability of resistance in these varieties. The molecular markers developed could be readily applied in marker‐assisted selection for MYMIV resistance genes in urdbean.