Isolating silkworm genomic DNA without liquid nitrogen suitable for marker studies

Abstract

Genomic DNA was isolated from posterior silk gland of silkworms, Antheraea assama. Absolute alcohol was used as tissue fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>40 kb). Samples yielded similar amount of DNA when fixed in absolute alcohol (400 μmg/g of silk gland tissue) and ground in liquid nitrogen (456 μmg/g of silk gland tissue). RAPD profile of the isolated DNA revealed high degree of polymorphism. The silkworms were analysed using 50 random primers among which 36 polymorphic primers gave 309 amplicons. The average amplicons per primer found to be 8.58 and 94.82% amplicons were polymorphic. Cluster analysis based on Jaccard’s similarity coefficients resulted in the formation of two main clusters with S9 on one cluster and the remaining strains on the other cluster. Jaccard’s similarity coefficients ranged from 0.122 to 0.863 indicating a high level of genetic diversity within muga silkworm collection. Isolated DNA was also suitable for cloning and restriction enzyme digestion. This method does not require liquid nitrogen for fixation, grinding or storage at -80°C, making it advantageous over other common protocols.

Key words: Genomic, silkworms, muga, molecular breeding.