Forensically informative nucleotide sequencing (FINS) for the first time authentication of Indian Varanus species: implication in wildlife forensics and conservation

Abstract

Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.

Visual detection of donkey-derived ingredients by loop-mediated isothermal amplification with 4-(2-pyridylazo)-resorcinol sodium salt

“…The visual LAMP method for detection of donkey-derived ingredients in common meat products was established in this study, and the detection limit was 1% donkey meat mixed into other species-derived meat. By comparison with other methods (ÅAkalar & Kaynak, 2016;Jia et al, 2016;Karabasanavar et al, 2017;Pebriana et al, 2017;Rajpoot et al, 2017;Sudjadi et al, 2016;Yuru et al, 2016), the developed LAMP assay had the advantages of cost effective, simplicity, high specificity, and sensitivity, which was suitable for surveillance of the commercial meat or meat products adulterated with donkey meat.…”

“…The food labels of meat and meat products are required to mark the meat source specifically to prevent adulteration in many countries, but the mixture of lower-price meat into high-price meat is still common, for example, adulterating pork or duck meat into beef or mutton or donkey meat, therefore, a reliable identification method of meat species is critical for surveillance of the commercial adulteration (Hong et al, 2017). The existing identification methods are mainly classified into protein-based approaches, for example, radioimmunoassay (Lowenstein et al, 2006), chromatography (Lozano et al, 2017;Pebriana et al, 2017;Wu et al, 2018) and Chemometrics-Assisted Shotgun Proteomics (Yuswan et al, 2018), and DNA-based approaches (Sheikha et al, 2017), for example, polymerase chain reaction (PCR) (Karabasanavar et al, 2017;Man et al, 2012;Mane et al, 2012), multi-PCR (Abuzinadah et al, 2015;Jia et al, 2016), realtime PCR (ÅAkalar & Kaynak, 2016;Herrero et al, 2013;Pegels et al, 2015;Sudjadi et al, 2016), PCR-RFLP (Bielikova et al, 2010), Biochip technology (Iwobi et al, 2011), forensically informative nucleotide sequencing (Rajpoot et al, 2017), and real-time PCR coupled melting curve analysis (Yuru et al, 2016). The structure of meat protein are usually destroyed by the processes such as shredding, cooking and roasting, therefore, the reliability of protein-based approaches is compromised, by comparison, the DNA-based approaches are more reliable, among which PCR is the most commonest assay, PCR, forensically informative nucleotide sequencing and melting curve for analyzing the adulterated meat product is very effective, but limited by the presence of PCR inhibitors in real biological samples and food samples, and meat products are the very complex matrix, mainly composed of proteins, lipids, pigments, enzymes, and other substances, which may interfere with PCR reactions (Wilson, 1997).…”

The preliminary molecular study of four skink species in Rajaji Tiger Reserve (RTR), Uttarakhand, using 12S rRNA mitochondrial locus

“…We set up cycling condition according to previously published condition (Rajpoot et al. 2016 ). On completion of PCRs, we electrophoresed PCR product on 1.5% agarose gel and visualized over transilluminator to detect the amplification.…”

“…Each reaction of 10 ll reaction contained 1 Â PCR buffer (50 mM KCl, 10 mM Tris-HCl and 2.5 Mm MgCl 2 ), 200 lM of each dNTP, 1.25 lg of BSA, 4 pM of each primer (forward and reverse) and 0.5U of Taq DNA polymerase (GeNei, Bangalore, India) and approximately 35-45 ng of genomic DNA. We set up cycling condition according to previously published condition (Rajpoot et al 2016). On completion of PCRs, we electrophoresed PCR product on 1.5% agarose gel and visualized over transilluminator to detect the amplification.…”

“…Although until there are no any molecular studies conducted on RTR skinks population, therefore here we did preliminary molecular study by using partial fragment of 12S ribosomal RNA (350 bp) (Kocher et al 1989) mitochondrial locus. Mitochondrial 12S rRNA mostly used for the species identification and deep phylogeny due to highly conserved characteristic and shows more difference between the interspecies (Kocher et al 1989;Kumar et al 2014;Rajpoot et al 2016).…”