DNA Tools to Establish Species and Individual Identity of Confiscated Biological Samples in Wildlife Related Crime : A Case Report

DNA techniques are prominently used in forensic investigations involving poaching and illegal trade of endangered animals. In the present case study, we could establish the link between crime scene and seized samples successfully using DNA markers. Three persons were caught, while trespassing the forest area near Badmalhera, Madhya Pradesh, India. Range forest officer seized skin and flesh of wild animal from these persons. The officer also found blood stains on stones and leaves at the suspected spot of crime. The samples seized from the accused and those collected from the crime spot were forwarded to our laboratory to identify species and to establish relatedness amongst the samples, if any. Species identification was done using universal primers’ for mitochondrial cytochrome b gene. All the forwarded samples were found to be of same species i.e., Indian spotted deer. Further, genotyping with eight polymorphic microsatellites revealed that the samples seized from the accused were similar to blood stains found at the crime spot.

The greatest challenges for enforcement agencies are to detect illegal poaching and trading of wildlife and to obtain concrete evidence for the same.DNA Forensics has become a major tool in wild life crime investigation [3].DNA analysis is not limited to only species identification but use of DNA for individual identification in wildlife forensic applications has also increased significantly [4].This involves investigation to determine if two confiscated samples are from the same individual, to determine the pedigree of a particular individual or for questions requiring assignment to population of origin [5,6].
In this case, both mitochondrial cytochrome b (cyt b) and nuclear microsatellites were used to establish the species and individual identity to link the evidences collected from the spot of crime and seized from the accused.

Case report
Three persons were caught while trespassing through the forest area at Badamalehra range in Madhya Pradesh.Skin and flesh of wild animal were seized from them.The investigating range forest officer also found blood stains on stones and leaves at the suspected spot of crime.Investigation team collected blood scrapings from the spot and forwarded them along with seized flesh and skin to our lab for species and individual identification.The case was registered as CCMB/WLFC-811 and the samples were given sample codes i.e., WL-1751 to WL-1754, where 'WL' stands for Wildlife.

DNA isolation
Blood scraping from the surface of the stones (WL-1751) and leaves (WL-1752) were directly transferred to 500 µl of blood lysis buffer [0.32 mM Sucrose, 10 mM MgCl 2 , 10 mM Tris HCl, 50 mM EDTA (pH 8.0), 30 µl of 20% SDS, 20 µl of 20 mg/ml Proteinase K].Piece of Flesh (WL-1753) and Skin (WL-1754) were given PBS (Phosphate Buffer Saline) wash for overnight to remove impurities like soil and dirt particles.After, complete washing, they were finely chopped and transferred to lysis buffer (50 mM Tris HCl (pH 8.0), 10 mM EDTA (pH 8.0), 100 mM NaCl, 30 µl of 20% SDS and 20 µl of 20 mg/ml Proteinase K).The samples in lysis buffer were briefly vortexed and incubated at 52 °C for 8 hrs with gentle rotation.After complete lysis, the samples were processed for DNA isolation using the conventional Phenol-chloroform method [7].The DNA was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and quantified using NanoDrop 2000 TM UV-Vis spectrophotometer.DNA quality was checked by visualizing it in 0.8% agarose gel.

Species identification
The DNA obtained from all the above samples was amplified using the 'Universal Primers' [8] to generate the species-specific molecular signature.The amplification was carried out in 20 µl reaction volume, using 20 ng of template DNA, 100 µm of each d NTPs, 5 pmol of each primer (Bioserve, Hyderabad, India), 1.5 mM MgCl 2 , 1 X of PCR buffer (10 mM Tris HCl, pH 8.3 and 50 mM KCl).The PCR conditions were: initial denaturation at 95 °C for 10 mins, followed by 35 cycles each of denaturation at 95 °C for 45 sec, annealing at 52 °C for 50 sec DNA Tools to Establish Species and Individual Identity of Confiscated Biological Samples in Wildlife Related Crime: A Case Report Abstract DNA techniques are prominently used in forensic investigations involving poaching and illegal trade of endangered animals.In the present case study, we could establish the link between crime scene and seized samples successfully using DNA markers.Three persons were caught, while trespassing the forest area near Badmalhera, Madhya Pradesh, India.Range forest officer seized skin and flesh of wild animal from these persons.The officer also found blood stains on stones and leaves at the suspected spot of crime.The samples seized from the accused and those collected from the crime spot were forwarded to our laboratory to identify species and to establish relatedness amongst the samples, if any.Species identification was done using universal primers' for mitochondrial cytochrome b gene.All the forwarded samples were found to be of same species i.e., Indian spotted deer.Further, genotyping with eight polymorphic microsatellites revealed that the samples seized from the accused were similar to blood stains found at the crime spot.
and extension at 72 °C for 1 min.The final extension was at 72 °C for 10 mins.PCR products were visualized in 2% agarose gel along with 100 bp DNA ladder (Invitrogen TM , USA) to confirm the correct amplicon size.All successfully amplified PCR products obtained were sequenced (ABI 3730) on both strands in triplicates and sequences were aligned using the program Sequencing Analysis 5.2 and Codon Code alligner.Statistical confidence of sequence similarity within and among species was further assessed using a Basic Local Alignment Search Tool (BLAST) [9].The sequence resolved was compared using MEGA 6.0 [10] with the molecular signatures of the vast number of known animal species (available in database of molecular signature generated and maintained by CCMB) and sequences of closely related Indian species available in mitobase of NCBI.All the aligned sequences for the cyt b showed the maximum sequence similarity with the cyt b sequence of Axis axis i.e., Indian spotted deer (Table 1).

Individual identification
After species identification, all the DNA samples along with one positive control (DNA of a known Indian spotted deer) and one negative control (No DNA) were subjected to PCR amplification using 8 microsatellite loci.All the 8 loci (Ca13, Ca18, Ca30, Ca38, Ca42, Ca60, Ca71 and Ca75) are deer-specific [11].PCR amplification was carried out in 20 μl of reaction mixture containing 30 ng of genomic DNA, 1XPCR buffer, 100 μM dNTPs, 1.5 mM MgCl 2 , 5 pmoles of each primer, and 0.5 units of amplitaq gold (Applied Biosystems, USA).The reaction conditions were: initial denaturation at 95 °C for 10 min, denaturation at 94 °C for 45 s; annealing at 51 °C to 61 °C for 50 s; and extension at 72 °C for 1 min (30 cycles) with final extension at 72 °C for 7 mins.Forward primers were fluorescence labeled.The PCR reaction was repeated three times and visualized in 2% agarose gel along with 100 bp DNA ladder (Invitrogen TM , USA) to confirm the correct amplicon size.All amplified PCR products were size fractionated and visualized on ABI 3770 DNA sequencer.The allele size was determined using Genemapper TM software version 3.1 (Applied Biosystems, USA).The multi-locus genotype profiles of all the samples were subjected to relatedness testing by calculating Likelihood Relatedness (LR) using ML-relate software [12].The multi-locus genotypes of other known individual Indian spotted deer were also used to compare the LR values.

Results and Discussion
In the present study, high molecular weight DNA was obtained from all the four samples namely WL-1751, WL-1752, WL-1753 and WL-1754.The cyt b sequences obtained for all four samples were aligned and compared with cyt b sequences of the vast number of known animal species in CCMB database and closely related Indian species i.e., Axis axis, Axis porcinus, Cervus unicolor, Antelope cervicapra and Boselaphus tragocamelus available in

Table 1 :
Comparisons of cytochrome b sequences generated from the seized samples with the sequences of reference animals of major Indian ungulate species involved in wildlife related crime in India.Only variable sites are shown.(Number at top represents variable positions).*Reference sequences from In-house database # Reference sequences obtained from NCBI Genbank (JN093092.1,AY035874.1,FJ556575.1,AF022058.1,and AJ222679.1)