Bruchid Resistance Study through Bulked Segregant Analysis: Used as a Preliminary Step for Next-Generation Sequencing

Abstract

Seed beetles, commonly known as bruchids, are serious insect pests of Indian pulses inflicting high damage during storage. Earlier, a bruchid resistant rice bean landrace (TNAU Red) from Manipur and a susceptible mung bean variety (VRMGg1) from Tamil Nadu were reported and their inter-specific cross was made in Tamil Nadu Agricultural University. In the present study, seeds from the segregating populations (recombinant inbred lines, RILs) of VRM (Gg)1 and TNAU Red were evaluated for bruchid Callosobruchus maculatus Fabricius resistance following standard methods under laboratory conditions. Bulked segregant analysis (BSA) is an efficient method for quick identification of molecular markers linked to any specific gene or genomic region. BSA significantly reduces the scale and cost by simplifying the procedure compared to conventional method of analyzing the entire population. BSA was carried out using 3 marker systems with an attempt to identify markers associated with the present trait of interest. Two DNA bulks namely resistant bulk (RB) and susceptible bulk (SB) were grouped by pooling equal amount of DNA from 5 each of resistant and susceptible RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP) and adult emergence from seeds. Marker analysis carried out with 41 polymorphic SSRs showed skewed segregation distortion towards the susceptible parent. However, out of 9 RAPDs and 10 ISSRs, 2 RAPD (OPB08 and OPX04) and 1 ISSR (UBC810) primers amplified an allele at 200 - 300 and 300 - 400 bp, respectively, in resistant parent and resistant bulk but not in susceptible parent and susceptible bulk. The amplification of resistant parent allele in resistant bulk may indicate the presence of bruchid resistant gene(s) in the resistant RILs. This experiment was aimed at verifying the presence of bruchid resistant lines carrying resistant gene(s) prior to further association mapping or QTL mapping studies. Further, such type of study can be applied as a preliminary step in NGS technology.
 HIGHLIGHTS
 
 Bruchids are the most damaging storage insect in pulses and other legumes causing great economic losses
 Bulk segregant analysis (BSA) conducted using three marker systems in an attempt to identify markers associated with bruchid resistance
 Categorization of RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP), adult emergence from seeds and scoring done
 Marker analysis using BSA useful for verification of the presence of bruchid resistant lines carrying resistant gene(s) prior to further advanced studies such as QTL mapping, NGS, etc.
 
 GRAPHICAL ABSTRACT