Genetic characterization of microsporidians infecting Indian non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) by using PCR based ISSR and RAPD markers assay

This study established the genetic characterisation of 10 microsporidian isolates infecting non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) collected from biogeographical forest locations in the State of Assam, India, using PCR-based markers assays: inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD). A Nosema type species (NIK-1s_mys) was used as control for comparison. The shape of mature microsporidian spores were observed oval to elongated, measuring 3.80 to $4.90{\mu}m$ in length and 2.60 to $3.05{\mu}m$ in width. Fourteen ISSR primers generated reproducible profiles and yielded 178 fragments, of which 175 were polymorphic (98%), while 16 RAPD primers generated reproducible profiles with 198 amplified fragments displaying 95% of polymorphism. Estimation of genetic distance coefficients based on dice coefficients method and clustering with un-weighted pair group method using arithmetic average (UPGMA) analysis was done to unravel the genetic diversity of microsporidians infecting Indian muga and eri silkworm. The similarity coefficients varied from 0.385 to 0.941 in ISSR and 0.083 to 0.938 in RAPD data. UPGMA analysis generated dendrograms with two microsporidian groups, which appear to be different from each other. Based on Euclidean distance matrix method, 2-dimensional distribution also revealed considerable variability among different identified microsporidians. Clustering of these microsporidian isolates was in accordance with their host and biogeographic origin. Both techniques represent a useful and efficient tool for taxonomical grouping as well as for phylogenetic classification of different microsporidians in general and genotyping of these pathogens in particular.