A Novel Microsatellite (STR) Marker for Forensic Identification of Big Cats In

Forensic Science International 141 (2004) 143–147

A novel microsatellite (STR) marker for forensic identiﬁcation of big cats in India Anju Singh, Ajay Gaur, K. Shailaja, B. Satyare Bala, Lalji Singh* Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India Received 12 June 2003; accepted 30 January 2004

Abstract

India is the home to five of the eight majestic big cats of the world. The three major big cats namely, lion, tiger, and leopard are listed in the Schedule I of the Indian Wildlife Protection Act, 1972. Apart from the severe loss of the habitat, these are continuously facing the danger of extinction mainly due to poaching and hunting for their body parts, which are being greatly valued by apothecaries marketing traditional Chinese medicines. With the advent of polymerase chain reaction (PCR), DNA-based markers have emerged as major tools in the arena of wildlife forensics. Microsatellites (short tandem repeats, STRs) are markers of choice because of their polymorphic and co-dominant nature. These strictly follow the Mendelian inheritance and are highly reproducible. We have identified a new microsatellite (STR) locus Ple 46, which shows amplification in a species- specific manner (size of STR) in all the members of the family felidae studied here. This PCR-based, non-invasive method opens a new avenue to forensic identification of big cats. # 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Big cats; PCR; Microsatellites; STR; Non-invasive; Wildlife forensics

1. Introduction 1000 poaching deaths every year, leopards could disappear soon from India by the end of the decade. India’s biodiversity is blessed with three major big cats of With the advent of polymerase chain reaction (PCR), DNA family felidae i.e. Asiatic lion (Panthera leo persica), Indian marker systems based on PCR have emerged as major tools or Bengal tiger (Panthera tigris tigris) and leopard (Panthera for various genetic analyses. Microsatellites/short tandem pardus). All these animals are listed in Schedule I of the repeats (STRs) are foremost among the molecular markers Indian Wildlife Protection Act, 1972. As with all animals used as standard tool for the investigation of forensic samples and plants, habitat loss is a severe cause of concern. But, due from human source and are applicable to trace evidences also to their high proﬁle in the eyes of poachers, hunters and [1–3]. Their main advantages are that they can be easily farmers, it is possible that big cats would disappear long assayed by PCR, they are expressed co-dominantly in phe- before their habitat does. They are large and dangerous and notype, and they are highly polymorphic in most organisms. their body parts are sometimes viewed as being of great Recently, there has been an increase in the forensic appli- medicinal value. The tiger is a walking apothecary according cation of STRs to cases involving non-human DNA, for to the traditional Chinese medicine. Parts of leopard can be example, wild animals hunted down by poachers, domestic easily mistaken for that of a tiger and are greatly valued by canines, felines, and even plants [4–7]. apothecaries marketing tiger medicines. Conservationists The objective of this study is to develop a simple and warn that at the present rate of attrition, with an estimated robust forensic method for identifying big cats, generally facing the problems of illegal hunting and poaching in India. Here we describe the development of a nuclear DNA marker * Corresponding author. Tel.: þ91-40-27160789; (microsatellite/STR) for identiﬁcation of various body parts fax: þ91-40-27160591. of big cats like lion, tiger and leopard using simple agarose E-mail address: [email protected] (L. Singh). gel and polyacrylamide gel electrophoresis (PAGE).

2. Materials and methods Gold DNA polymerase (Perkin-Elmer) in a 20 ml reaction volume. Amplification was performed on a thermal cycler 2.1. Sample collection (M J Research, USA). The reaction conditions were: initial denaturation at 94 8C for 10 min, denaturation at 94 8C for A total of 88 wild-caught individuals, housed in various 30 s; annealing at 57 8C for 30 s; and extension at 72 8C for zoological parks were sampled in this study. Blood samples 45 s (30 cycles) with final extension at 72 8C for 7 min. of Asiatic lions (P. leo persica), hybrids between Asiatic and For hair and faecal samples the reaction conditions were the African lions (Panthera leo leo), Indian or Bengal tigers same except the total number of cycles increased to 40. (P. tigris tigris), and leopards (P. p ard us) were collected from The PCR products were electrophoresed on a 3% agarose the femoral vein by immobilizing the animal in squeeze cages gel, stained with ethidium bromide (0.5 mg/ml) and photo- or by anesthetizing them. In addition to blood samples, hair, graphed. and faecal samples were also collected from some individuals. Bone, skin, and meat samples sent for forensic identi- 2.5. Genotyping fication were also included in the present study. For microsatellite analysis, the forward primer was fluor- 2.2. Isolation of DNA escence-labeled and the PCR amplification was carried out using same conditions as described earlier. The fluores- DNA was extracted from the whole blood using standard cence-tagged PCR products were size-fractionated on dena- phenol:chloroform method [8]. DNA from hair samples was turing 6% polyacrylamide gel and visualized on ABI 377 isolated using the protocol of Ohhara et al. [9], from faecal DNA sequencer (Applied Biosystems, USA). The allele size samples using the protocol of Shankarnarayanan and Singh was determined using GENESCANTM and GENOTYPERTM [10], and from skin and meat tissues using the protocol software (Applied Biosystems, USA). Heterozygosity level reported by Fang and Wan [11]; DNA from bones was was estimated using ARLEQUIN software [15]. extracted using a modified protocol of Yue and Orban [12]. 2.6. Cloning and sequencing 2.3. Identification of Ple 46 In order to sequence the Ple 46 locus in different species Partial genomic library of Asiatic Lion was constructed of big cats, an aliquot of the PCR product was ligated into and screened with various di-, tri-, and tetra-oligonucleotide pMOS Blue vector using pMOS Blue Blunt End Cloning kit repeats [13]. Ple 46 (accession number AY095489) is one of (Amersham Biosciences, UK). Five recombinant clones the 31 polymorphic microsatellite markers characterized in from each individual were sequenced using U19 and T7 Asiatic lions. It is a highly conserved microsatellite locus universal primers and automated ABI 3700 DNA sequencer having the repeat motif (CA)22. Primers were designed (Applied Biosystems, USA). Sequences were aligned using from the sequences flanking the microsatellite repeat using software like AUTOASSEMBLER (Applied Biosystems, the software Primer 3[14]. The forward primer used was USA) and CLUSTALX 1.8 [16]. 50-GAGGACGGTCTGGTGGAGT-30 and the reverse primer was 50-AACTTTAACCCACTGCTGCC-30. 3. Results 2.4. PCR amplification The microsatellite locus Ple 46 was successfully amplified PCR was performed with 50 ng of genomic DNA, 5 pmol using DNA isolated from different samples in all the species of the primer, 200 mmol each of dATP, dGTP, dCTP, dTTP, 1 of big cats. The PCR products were resolved on 3% agarose Â PCR buffer containing 1.5 mM MgCl2, 0.1 units of Taq gel (Fig. 1). PCR products varied in size considerably in the

Fig. 1. Distinct band pattern of the STR locus Ple 46 amongst big cats as visualized in 3% agarose gel. A. Singh et al. / Forensic Science International 141 (2004) 143–147 145

Fig. 2. Electropherogram showing some of the alleles at the STR locus Ple 46 in different members of family Felidae. three species of big cats with lions having the largest From the above experiments it is quite obvious that Ple 46 followed by leopards and tigers, respectively. The size is not very polymorphic in big cats with only four alleles difference was clearly visible in the agarose gel. To resolve observed in Asiatic lions, four in hybrid lions, two in tigers, the products further and to ascertain the allelic size, these four in leopards, and two alleles (89–91 bp) in domestic cats products were loaded in 6% polyacrylamide gel. The elec- (Fig. 3). However, observed heterozygosity level at locus Ple tropherogram also revealed clear-cut variation in size among 46 was >75% in all the three big cats. the three big cats (Fig. 2). PCR products obtained from hair, Cloning and sequencing of PCR products from different faecal, skin, meat, and bone samples also gave the similar species of big cats under investigation further revealed that allelic profiles as those from the blood samples. Initial lions (from which Ple 46 was isolated) have the longest studies were carried out with 2–3 individuals from each uninterrupted stretch of CA repeats i.e. (CA)22. The size of of the three feline species; but to further confirm the range of CA repeats in leopards was (CA)14–15 followed by those in alleles in these species, a total of 88 individuals were tigers, which have the shortest and interrupted (CA)7–8. The screened. 20 Asiatic Lions and 15 hybrid lions were screened CA repeats in tigers were interrupted twice by A ! G to determine the allele size range in Lions, which was found transitions at second and fourth position in repeat motifs. to be 111–119 bp. In addition to this, DNA from 35 leopards Domestic cat has a longer and uninterrupted stretch of CA and 18 tigers were also PCR amplified, and the allelic size repeats i.e. (CA)10 (Fig. 4). range was found to be 97–103 and 83–85 bp, respectively. Ten domestic cat (Felis catus) samples collected from different places were also included in the study to ensure 4. Discussion the feline-specific nature of this STR locus. Efforts to amplify this locus in wolves, dogs, deers, and cattle yielded Asiatic lions, confined to a single location in wild, in the no amplification. Gir Forest in Gujarat state of India, are the focus of special 146 A. Singh et al. / Forensic Science International 141 (2004) 143–147

Fig. 3. A graphic representation of distribution of alleles at STR locus Ple 46.

Fig. 4. Sequence alignment at STR locus Ple 46, in major feline species of India. Only two representative samples from lion, tiger, and leopard have been shown to exemplify the varying lengths of repeat motifs. (*) The numbers represent the location of forward and reverse primers. conservation efforts and are highly protected. Most of the having the longest repeat and the primitive one having the forensic cases in India involving big cats are either of tigers shorter repeat length. or leopards. The Wildlife Protection Society of India has Amplification of STR locus Ple 46 in all the members of cat documented the killing of 61 Tigers and 188 Leopards, since family including the big cats suggests that it is a highly January 2002. The actual number is estimated to be at least conserved locus (unpublished data). Homologues have been seven times higher, since a majority of wildlife crimes go found between several species, which diverged more than 470 unreported. This PCR-based method can provide material million years ago [20]. Conservation of flanking sequences evidence to wildlife enforcement and judicial organizations. including the priming sites suggests that selection must be It involves amplification of locus Ple 46, a CA-rich micro- operating conservatively at this locus. Homology of flanking satellite marker developed from Asiatic lion, P. leo persica sequences to various ESTs like Mus musculus cDNA clone [13]. and human brain cDNA clone further suggests that the The stretch of CA repeats at locus Ple 46 was the longest microsatellite repeat region is probably embedded in the in lions (CA)22, from which it was originally identified. intron of a protein-coding gene as has been observed in sharks, Microsatellites may be consistently longer in original spe- where locus Loc 6 repeat motif and flanking sequences have cies just because of the clone selection process, something been found to be conserved since 250 million years [21]. known as ascertainment bias [17,18]. Tigers have the shortest and highly interrupted stretch of CA repeats. This obvious difference in repeat length between these three 5. Conclusion species of felids is consistent with the hypothesis of Zhu et al. [19], where they proposed that increase in length is DNA markers already available to identify big cats involve more likely in microsatellite, with the most evolved species sequencing of mitochondrial region or other species-specific A. Singh et al. / Forensic Science International 141 (2004) 143–147 147 nuclear marker, which is very laborious and time consuming. [7] S. Gilmore, R. Peakall, J. Robertson, Short tandem repeat Here we report a simple PCR-based method for identifica- (STR) DNA markers are hypervariable and informative in tion of big cats and their parts using a novel microsatellite Cannabis sativa: implications for forensic investigations, locus, Ple 46. The most important aspect of this marker is Forensic Sci. Int. 131 (2003) 65–74. [8] J. Sambrook, E.F. Fritsch, T. 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