Identification of animal species through reminiscent biological material
Abstract
Poaching and illegal trade in wild animals and their products are highly detrimental for wildlife biodiversity and ecological balance. Therefore, it is important to effectively implement the existing laws pertaining to wildlife protection. For efficient implementation of such laws, wildlife protection agencies must have validated quick, easy and reliable tools for the identification of wild animals from different type of samples such as skin, feather follicles, faeces, tissue and bone. As forensic experts many a times face such situation in which samples are presented in degraded, old condition and scant quantity. So, there is also a need to develop suitable and reliable protocols for identification of species from such samples. Keeping in view, the depleting populations of wild animals especially important animals of food chain like Tiger (top predators), black buck and symbols of our cultural heritage such as peacock and all other perspectives, a study for developing protocols for identification of species from different kinds of samples and further differentiation of wild species from closely related species has been carried out. Several quicker and non–hazardous protocols such as chelating resin extraction method and CF11 extraction method were standardized for DNA extraction from different kinds of old, degraded and other samples collected from field. The genomic DNA extracted was quantified using high fidelity Picodrop™ UV spectrophotometer. Several old samples collected in 2006, from peacock, blackbuck and deer were used in the study to extract DNA and extracted DNA was successfully amplified by PCR/ Real time PCR. The samples yielding amplifiable amount of DNA were amplified using different sets of universal and species specific primers. Universal primers targeting 12S rRNA were designed during the study to amplify DNA extracted from Avian spp. samples. Species specific primers targeting D-loop mitochondrial gene of tiger were used to amplify the DNA extracted from tiger blood samples. DNA extracted from different samples were also amplified using cyt b gene targeting universal primers. A 12S rRNA gene based PCR-RFLP method
was standardized to differentiate between peacock and chicken. The differentiating enzymes such as Alu I and Mbo II were used. On digestion of PCR product, AluI yielded two products in case of peacock while three in chicken. However, Mbo II was yielding a clear difference between the two species, giving two products in case of peacock while it had no cut site in chicken. These results were confirmed using in silico RFLP analysis of the sequences obtained after nucleotide sequencing. Hence, PCR-RFLP was found to be of value in differentiating between such closely related species. The amplified products obtained by using different sets of primers such as cyt b gene products of neelgai, black buck, D-loop gene species specific products of male and female tiger and 12S rRNA gene products of peacock and chicken were cloned and sequenced. In order to determine that there is no cross amplification and to know the Forensically Informative Nucleotide Sequence (FINS), the sequence analysis was done. The sequence analysis revealed several facts that were found to be supported with the already documented data. From the sequence analysis of black buck sequence used in the study (cyt b gene) it was found to be closer to blackbuck from Japan and France. Sequence analysis of D-loop gene specific sequence of female tiger revealed that it does not belong to Panthera tigris tigris (Bengal tiger). However, this fact was confirmed and further made clear when the sequencing of cyt b gene PCR product of female tiger was done. The sequence analysis revealed that the female tiger belonged to Panthera tigris jacksoni (Malayan tiger), thus confirming it to be of different breed. Sequence analysis of sequence obtained from peacock was also done and the peacock in the study was found to be closer to peacock of India and was grouped with other peafowls such as Pavo cristatus of USA and Pavo muticus on phylogenetic tree analysis. Sequence analysis of neelgai cyt b gene sequence revealed it to be closer to Neelgai of India and species Tetracerus quadricornis of deer family. Thus, the origin of samples was identified/confirmed using various confirmatory tools such as sequencing and PCR-RFLP. The samples in which end point PCR was not able to yield any appreciable result was further amplified and quantified using real time PCR and the assay was able to detect DNA amount upto 21.4 pg/μl. The study conducted paves the way for quick and easy identification of species from various kinds of forensic samples obtained from field