Development of molecular tools to differentiate Indian wild pig ( Sus scrofa cristatus ) meat from exotic and local domestic pig meat

Aim: Identification of wild pig and domestic pig is essential to prevent illegal poaching of wild pig and to implement Wildlife (Protection) Act, 1972. PCR-RFLP was used to differentiate Wild pig (Sus scrofa cristatus) from Domestic pig (Sus scrofa domestica) meat. Materials and Methods: DNA was isolated from meat samples of both the sub species and a fragment of Cytochrome b gene was amplified using universal primers and the PCR products were subjected to restriction digestion. Results: All the known samples of each of the sub-species amplified 474 bp fragment successfully using b1 and b2 primers. To differentiate between wild and domestic pig meat, restriction digestion of the PCR products was carried out to produce characteristic PCR-RFLP patterns for each species. StuI digestion yielded a RFLP pattern which distinguished the closely related sub species. The alignment of sequences of Wild pigs with sequences of local domestic pig, European wild pig and exotic breeds revealed 7 intra-species polymorphic sites within Cytochrome b gene fragment. Conclusion: This study showed that The PCR-RFLP is a simple and very effective tool for differentiating the samples of both the sub species and could prove to be a useful tool in forensic identification of wild pig and domestic pig.


Introduction
mitochondrial DNA (mtDNA) has been used for these studies [3][4][5][6][7][8][9].Due to maternal inheritance of mtDNA, Indian wild pig (Sus scrofa cirstatus) is a protected no recombination mechanism exists as in the nuclear species under Schedule III of the Indian Wildlife DNA to eliminate error once a mutation occurred [10].(Protection) Act, 1972 and a separate sub-species from Thus, accumulation of these point mutations allows the domestic pig (Sus scrofa domesticus), which is a discrimination of closely related species [11].However, source of meat and an important farm animal in many attempts on within species or closely related substates of India [1].Indian wild pig is extensively species identification using simple PCR-RFLP method poached for meat and in the absence of simple are scanty.differentiation protocol for meat identification of wild The cytochrome b gene for species identifications and domestic pigs, implementation of Wildlife have been used by many researchers as it is one of the (Protection) Act, 1972 remains a constraint.bettered genes in the genbank and has superior ability Most of the methods used for identification of for separating species when compared to other genes species of origin of meat have been reported to have [12].In the present study, the partial sequence of the limitations in use due to problems in specificity (i.e.[2].DNA-based methods for meat identification Five known samples (meat) of each domestic and include Polymerase Chain Reaction [3], Polymerase wild pig collected from fresh carcass were used for Chain Reaction-Restriction Fragment Length DNA isolation using DNeasy Blood & Tissue Kit Polymorphism (PCR-RFLP) [4,5], Random Amplified (QIAGEN, Germany) in a final elution volume of 300 Polymorphic DNA (RAPD) [6], Single Strand µl.The DNA was subjected to PCR amplification in a Conformational Polymorphism (SSCP) [7] and displacement loop region (D-loop) [20] have been 5 CCAATGATATGAAAAACCATCGTT 3 and b2-5 ' used for species identification.GCCCCTCAGAATGATATTTGTCCTC 3) were used All the known samples of each of the sub-species for PCR amplification [13].The amplified product was amplified 474 bp fragment successfully using b1 and subjected to electrophoresis on 1.2% agarose gel for 45 b2 primers.Jon et al. [21] have also used this primer in min at 70V in Tris Acetate EDTA (TAE) buffer and gel the ARMs PCR to determine the presence of tiger bone was stained with ethidium bromide (Et br) (0.5µg/ml).
in Traditional Chinese Medicine (TCM).We validated The amplification parameters were 95°C for 20 the sequence authenticity by direct sequencing of one minutes followed by 35 cycles of 95°c for 1 min, 50°c PCR product each of Indian Wild pig and domestic for 1 min and 72°c for 1min with final extension at local pig.Together with these sequences, a total of 8 72°C for 10 minutes prior to a 4ºC hold.
mitochondrial sequences of Cytochrome b gene One PCR product of each subspecies were fragments were aligned to discern intra-species purified and sequenced in both directions using the variations.Seven polymorphic sites were identified in BigDye® sequencing kit (Applied Biosystems, Foster the sequence of Indian wild pigs and sequences of City, California, USA).The obtained sequences have samples collected from Western Ghats.Based on the been submitted in Gene Bank under Accession No.
intra-species variations, the Indian wild pig were KF185090 and KF185091.domestic pig, European wild pig and exotic breeds engyme, 17µl nuclease free water and incubated at th (Plate-1).The presence of 5 polymorphic site ("C") at 37ºC for overnight.The digested products were the position of 14466 (Table-1) created an additional subjected to electrophoresis on 1.5% agarose gel at 1.5 restriction site for StuI.V/cm for 4 hours and stained with Et br.Band patterns Apart from these polymorphisms, at the position were analyzed by Gel documentation System of 14370 and 14407, all the sequences of Indian origin (AlphaDigi Doc RT, JH India Ltd).
had "C" instead of "T" in European wild pig, Duroc

Results and Discussion
and Hampshire pig breeds.Due to the relatively high copy number of Srilankan wild boar (Sus scrofa affinis) was mtDNA genomes per cell, mtDNA is more suitable differentiated from native village pig (Sus scrofa than nuclear genome for studies on degraded or domestica) targeting two polymorphic sites of otherwise compromised materials [15].Various genes mitochondrial D loop and restriction digestion with while PCR-RFLP pattern of two other pieces of seized sequenced previously to study the phylogenetics of the meats under the same case matched with the domestic two sub species [1].Though the DNA sequencing pig profile (Figure -2).revealed unambiguous result, PCR-RFLP of 474bp Cytochrome b gene fragment could prove a quick and cost effective tool in forensic analysis of seized samples.The recent introgression of exotic genome due to the state sponsored programs conducted to upgrade the local population with imported semen or live animals of exotic breeds has diluted the gene pool of local breeds and precise breed specification of the local domestic remains questionable.In most of these upgrading programs paternal and not maternal introgression is possible as it is the imported semen or boar that is used commonly in the breeding program.Owing to maternal inheritance, polymorphisms of mitochondrial genes provide means in species identification.The polymorphism observed in In comparison to other phylogenetic marker, the Using the PCR-RFLP assay, we could resolve a cytochrome b gene demonstrates greater congruence forensic case registered under Damoh range, Madhya Three additional sequences discriminated by restriction digestion with StuI which of pig samples collected from forests of Western Ghats, generated bands of less than 200 bp (two 142 bp and one sequence each of European Wild pig, Hampshire one 190 bp bands) while two bands of 290 bp and 184 and Duroc breed were retrieved from National Centre bp were observed for Indian wild pig and domestic pig, for Biotechnology Information (NCBI).The sequences respectively, on 1.5% Agarose gel (Figure-1).Due to were aligned using DNAstar program (DNAS Inc, overlapping of bands of 142 bp only two bands (142 bp Madison, WI, USA).and 190 bp) are visible on restriction digestion with For differentiation, Cytochrome b gene fragment StuI in wild pig.The RFLP patterns of StuI on sequences were analyzed by DNAStar software and ↓ amplified PCR product were highly specific.The StuI restriction enzyme (AGG CCT) was used for sequence analysis of all the known samples of Indian RFLP analysis.The amplified PCR products were wild pig revealed two StuI sites, while only one site of restriction digested with StuI [14] in 30µl volume restriction was present in the amplified sequence of containing 3 µl 10X buffer, 10µl of amplicon, 1µl