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

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.