Identification of microsatellite markers linked to thermotolerance in silkworm by bulk segregant analysis and in silico mapping

Silkworm, being a poikilothermic insect, its growth and development is affected by environmental factors especially, temperature. In tropical countries like India, it has considerable effect on silk production due to the prevailing of hot climatic conditions. Previous attempts to evolve silkworm breeds and hybrids tolerant to high temperature by traditional breeding methods have not yielded the desired results. Hence application of new strategies like marker assisted selection (MAS) could be the most effective strategy for developing a thermo-tolerant bivoltine silkworm for sustainable silk production in India. As a prelude, in this study it is aimed to identify simple sequence repeat (SSR) markers closely linked with thermotolerance in silkworm. To do so, 20 silkworm breeds were evaluated at high temperature (36°C) and based on pupation percentage, two multivoltines (Nistari and Cambodge) and two bivoltines (SK4C and BHR3) were identified as thermo-tolerant and one bivoltine (CSR2) was identified as the susceptible breed. These breeds were screened with 85 SSR markers drawn from different linkage groups and out of those, only 11 markers (12.9%) showed distinct polymorphism between thermo-tolerant and susceptible breeds. Further, bulked segregant analysis (BSA) was performed using 11 polymorphic SSR primers, by comparing the SSR profiles of the tolerant (Nistari) and susceptible (CSR2) parents, their F1 and F2 bulks. Nevertheless, only 5 markers generated clear differences in the amplified DNAs between the bulks corresponding to that of the parents suggesting that the DNA regions amplified by these SSR markers are closely linked to thermotolerance in B. mori. The results obtained through bulk segregant analysis was further confirmed by genotyping 5 linked SSR markers using 140 individual F2 progenies. Of these 5 markers, highest Spearman's rho correlation coefficient was shown by S0816 indicating a high degree of closeness between the genotypic and phenotype variations in F2 population. Furthermore, we have also attempted to locate the genes near to S0816 by in silico approach and upshot revealed 3 genes nearer to its sequence on the B. mori genome. The BGIBMGA005249 gene was found to be located nearest to S0816 at a distance of 14.8 Kb. But, further studies are required in this regard to derive a relationship between the thermotolerance and the functional role of identified genes nearer to the closest marker, so that the identified markers can be used to develop a thermo-tolerant silkworm breed through MAS