Molecular Identification of Captive Snakes from their Shed Skin Using Cyt b & 12s rRNA Markers

Aims: To successfully utilize non-invasive shed skin samples for species identification of captive snake species of Tamil Nadu and create genetic repository. Study Design: The experiment was designed to apply the ammonium acetate method of DNA extraction from shed skin. Two mitochondrial markers were used to ascertain identification of species. Place and Duration of Study: Advanced Institute for Wildlife Conservation (AIWC), Tamil Nadu Forest Department, Vandalur, Chennai, Tamil Nadu. The samples were collected between May and October 2019 from Arignar Anna Zoological Park, Vandalur, Guindy National Park, Chennai, and Amirthi Zoological Park, Vellore, Tamil Nadu. Methodology: We collected fresh shed skin from 8 different snake species from captivities of zoos and dried between 48 to 72 hours to remove moisture. Independent DNA isolations were performed for each sample. The DNA isolated samples were quantified using Nanodrop Spectrophotometer for concentration. Independent PCR amplification of mitochondrial regions of cytochrome b and 12S rRNA were performed and agarose gel electrophoresis was carried out. PCR products were subjected to sanger sequencing using genetic analyzer. Results: The DNA concentration from all 8 different snake species ranged between 250 to 1600 ng/µL and average quality ratios A260/280 of 1.85 and A260/230 of 2.10. Both the mitochondrial gene regions cytochrome b and 12S rRNA showed specificity in species amplification with NCBI BLAST result ranging from 99-100%. Phylogenetic trees using maximum-likelihood method classified closely related species under the same clade, with a bootstrap support of 60-100%. Genetic distances of snake species ranged from 0.148-0.457 in cytochrome b region and 0.148-0.457 in 12S region. Conclusion: Shed skin is often overlooked from utilization for species identification. In this study, DNA from shed skin of 8 captive snakes is extracted and amplified using Cyt b and 12s mitochondrial markers. Individual phylogenetic trees are constructed for each marker to find relatedness of different snake species with one another. This work is an initiation of genetic repository creation of captive snake species of Tamil Nadu and could be effectively employed in conservation and population genetic studies of snakes.