ABSTRACT

The study is aimed at establishing a standard method for the isolation of Microsatellites from the wild silkmoth Antheraea mylitta, ecorace Daba trivoltine. Microsatellites were isolated by the selective hybridization method in which DNA fragments containing repeat motifs specific to repeat probe used in hybridization were captured.

In the present study six biotinylated oligos of the Di-, Tri- and Tetranucleotide repeat types were used as probes in individual hybridization reactions. Genomic DNA was digested with Sau3A, dephosphorylated, size selected from 100-1000 bp to which Sau3A ds linkers were ligated. The linker ligated genomic digest was hybridized against biotinylated repeat oligo and eluted microsatellite enriched DNA fragments were subjected to linker digestion with Sau3A, purified and cloned into BamH I digested pBluescript (KS+) plasmid and transformed into INVaF competent cells.

Totally 110 white colonies were screened and the clones having insert size above 100 bp were selected and sequenced. Totally 81 clones were sequenced of which 18 were found to be positive for microsatellites. Out of 18 microsatellites 15 are dinucleotide repeats and 3 are trinucleotide repeats and no tetranucleotide repeats were captured.