Application and Validation of Molecular Markers Linked to Yellow Mosaic Virus Disease in Mungbean (Vigna radiata (L.) Wilczek)

Abstract

Green gram or mungbean (Vigna radiata L. Wilczek) is one of the most widely cultivated crop in India. Mungbean Yellow Mosaic Virus(MYMV) belonging to the genus begomovirus causes the yellow mosaic disease in Mungbean (Vigna radiata). The objective of this study is to validate the molecular marker linked to MYMV resistance genes for rapid identification of resistant genotypes. 35 genotypes of Mungbean were also subjected to DNA fingerprinting and were evaluated for resistance to MYMV. Eight molecular markers including 5 SCAR markers and 3 SSR markers reported to be linked to YMV resistance in mungbean were validated for their usefulness in marker assisted breeding. Only six markers (MYMVR-583, CYR1, SCAR ISSR811 (YMV1), CEDG-198, DMB-SSR182 and DMB-SSR 186) produced distinct and reproducible bands of corresponding size and were found polymorphic between resistance and susceptible mungbean genotypes studied and none of the three SSR marker was found to be linked with YMV resistance gene. MYMVR-583, YMV1 and CYR1 markers are partially linked to MYMV resistance with the possibility, MYMVR-583 and YMV1 marker locus are primarily responsible for MYMV resistance, while CYR1 marker locus provides an epistatic interaction conferring superior quality like stable MYMV resistance. The band corresponding to MYMVR-583 gene was eluted and cloned in pTZ57R/T vector which was followed by confirmation using colony PCR, plasmid DNA isolation and Restriction digestion approaches and subjected to sequencing and were further subjected to in-silico analysis. The sequence data was validated by BLAST analysis and phylogenetic tree was constructed using MEGA5.0 software which revealed close similarity of sequence with other virus resistance genes. All the 35 mungbean genotypes were also examined for biodiversity analysis using 15 ISSR markers. 41 bands were detected with variable degree of polymorphism generating 78.33% of polymorphism with an average of 2.73 bands per primer. The size of fragments ranged from 110 bp to 1350 basepair. Number of amplified fragments with ISSR primers ranged from 1 to 6. PIC value ranged from 0.00 (UBC 810, UBC 812 and UBC 844) to 0.705 (ISSR 8US) with a mean of 0.3676. Resolving power varies from 1.656 (UBC 842) and 8.398 (UBC 813) with an average value of 3.58. ISSR primers ISSR 9, UBC 857, ISSR 01 having above average PIC and RP were more efficient and ISSR 8US was most polymorphic. The Jaccard’s similarity coefficient for the ISSR data set varied from 0.366 to 0.976 with a mean of 0.671. The Jaccard’s similarity indices based on ISSR profiles were subjected to UPGMA cluster analysis and genotypes grouped in five major groups. 21 out of 35 released cultivars grouped to cluster III. This indicated the narrow genetic base in the Indian mungbean cultivars used in the study. The details of diversity analysis and possible reasons for narrow genetic base in mungbean cultivars are discussed in the present study.