Genotyping of species has commonly been used to study their population genetics and to ascertain the source in poaching cases using non-invasive methods. We collected putative tiger scat samples from different tiger reserves. Nearly 100 tiger scats were identified based on morphology and collected from the Pakke Tiger Reserve in three months. Primarily DNA (>10Kb) was extracted from reference tiger blood samples using EZ1 DNA blood kit (Bio Robot® EZ1, QIAGEN, Germany) to optimize protocols for identification of the species, sex and genotyping, In case of scats sample, there is need to analyze whether the selected scat belongs to felids or any other species. Therefore, PCR conditions were optimized for amplification of different sizes of fragments (˜250bp, 381bp, 471 bp, ˜550bp) of mitochondrial cytochrome b gene, 16S rRNA gene and D-loop region using several primers with reference DNA. In view of poor quality (300bp to 500bp) of DNA from scat samples, a primer pair was designed to amplify the short fragment of mitochondrial cytochrome b gene, specifically of tiger and leopard. The RFLP profile will subsequently be developed to differentiate tiger and leopard. In order to identify sex from scat, we have optimized PCR conditions using primers (Goyal et al., unpublished) for sex specific molecular marker of zinc finger protein (ZFX/ZFY), yielding two short fragments (X-specific, 132bp, Y-specific, 197bp). Different reference DNA samples viz. Sus scrofa (n=2), Boselaphus tragocamelus (n=1), Bos taurus (n=1), Axis axis (n=2), Cervus unicolor (n=5), Panthera tigris tigris (n=2), Panthera pardus (n=1), were analyzed to answer whether there is any cross amplification with prey species or not, using this primer. After several trials, it was concluded that this sex specific primer amplifies DNA of Cervus unicolor. Therefore, primers will be redesigned to use with scats. DNA was extracted from 30 different scat samples (probably of tiger) of the Corbett Tiger Reserve using QIA amp® DNA Stool Mini Kit (QIAGEN, Germany). Success rate of DNA extraction from scat samples was recorded 90% by visualizing on 0.8% agarose gel. Standardized PCR conditions for mitochondrial DNA (cytochrome b gene, 16SrRNA gene and D-loop region), resulted in 98% success in amplification of DNA extracted from scats. We optimized PCR conditions for different microsatellite loci of tiger DNA samples. These standardized conditions will be applied to create genotype profile of tiger population using scats collected from different tiger reserves.

Dr. S.P. Goyal.