The use of DNA testing with regard to wildlife crimes has centred primarily on mitochondrial DNA (mtDNA). mtDNA in forensic investigations have been the subject of several studies [1, 5, 10, 12, 26]. These reviews have focussed on the use of mtDNA for the purposes of human identification. The advantages of using mtDNA in human identification are also true for wildlife studies. Samples frequently encountered in forensic wildlife studies are poor in nature and may have suffered much environmental insult. The large number of copies of mtDNA in every cell, with up to 10,000 copies of mtDNA per cell [6], is of much use when analysing degraded samples. Equally the protection afforded by the protein coat assists in the persistence of mtDNA. The mutation rate in mtDNA is known to be up to 10 times higher than that of nuclear DNA due in part to the low fidelity of mtDNA polymerase and the lack of a repair enzyme [7, 17]. This higher mutation rate has been used in phylogenetic studies of the human race [26] and for the same reason plays a part in taxonomic studies.

The mitochondrial genome in higher vertebrates is highly conserved in sequence ranging from 13 – 19 Kb [8, 15, 19]. The order of the genes shows little divergence with all higher vertebrate mitochondrial genomes encoding 37 genes. Twenty two of the genes encode tRNA molecules which are part of the mitochondrial protein synthesis process. Two genes are present that encode the small (12S) and large (16S) subunits of the mitochondrial ribosomal RNA. The gene cytochrome b (cytb) is a subunit of the cytochrome c oxidase and is the subject of much interest in taxonomic studies.



For a DNA locus to be of value in species identification it must show a high degree of inter species variation such that two closely related species can be distinguished. The locus should also exhibit little intra species variation such that members of the same species can be identified. There are a few loci on the mitochondrial genome that meet these requirements sufficiently such that they have played leading roles in taxonomic studies. The cytb locus is probably the best-studied locus for species identification along with the 12S RNA gene [2, 4, 9, 13]. The cytb gene is approximately 1140 bases in length and encodes a protein of 380 amino acids in size. In human mtDNA it is between bases 14,747 and 15,887. For other mammals the number of the base varies but the relative position of the locus is the same being close to the displacement loop and hypervariable region 1. The sequence of this gene from representatives from all the mammalian families and numerous other species has been deposited on the EMBL (www.ebi.ac.uk) or GenBank® (www.ncbi.nih.gov) databases.

The 12S rRNA gene on the mitochondrial genome, from position 650 to 1603 in the human mitochondrial map, meets the same requirement for species identification as the cytb gene. Universal primers [16] were developed to amplify a fragment of 456 bases on the cytochrome b gene for most mammalian species [11].

The most common method of species identification is to amplify all or part of the gene such as the cytb or 12S rRNA and directly sequence the PCR product. The resulting DNA sequence is compared to the DNA sequences on the EMBL and GenBank® DNA databases. Such comparisons using Basic Alignment Search Tools (BLAST) will display the closest alignment and also sequences with less homology. A complete match between any unknown and a lodged sequence on the database can lead to identification. Case reports have been published for the identification of tiger [24] panther [23] deer [28] snakes [27] bear [3] game birds [21] and rhino [14].

The degree of conservation of sequence using the cytb gene is sufficient to discriminate between closely related species. Wolf (Canis lupus) and the domestic dog show only a very few differences, 4 bases out of the entire gene. It is thought that dogs arose from the domestication of the wolf around 15,000 years ago [20]. This short divergence in time has led to few polymorphisms arising. Wolf and dog are classified in the same genus (Canis). The common red fox of Europe (Vulpes vulpes) is of a different genus (Vulpes) but same taxonomic family (Canidae). The red fox and the wolf show significant variation at the cytb locus as expected but exhibit greater homology when either species are compared to the domestic cat (Felis catus); although all the dog, wolf, fox and cat are all members of the taxonomic order (Carnivora). This illustrates how the genetic evidence can support previous taxonomic listings based upon physical features.

When the entire cytb locus for different species is aligned it is evident that there are regions of homology between all members of a family with a few domains exhibiting greater diversity. This leaves open the possibility of using ‘universal’ primers in conjunction with species specific primers. This type of approach has been used to identify the presence of tiger (Panthera tigris) bone from traditional Chinese medicines (TCM) [25].  The benefit of this type of technique is that it does not need to amplify a large segment of the cytb gene and then directly sequence the PCR product. From TCM samples which may be highly powdered it may be that the DNA is too degraded to permit amplification of a large segment of the gene. In such cases it may be possible to target a few bases known to be polymorphic between species. This approach, not unlike minisequencing used in mtDNA typing for human identification [22] is a step toward single nucleotide polymorphisms (SNP) testing. A range of SNP sites can be identified for many species with the cytb locus, or other loci found on the mtDNA such as the 12 S RNA, to allow species specific testing using a number of SNP primers. Provided sufficient SNP loci are used a particular species can be identified as being present.  This has applications for the food industry rather than the forensic community but such methods may be applied to animals on the CITES appendices rather than domesticated animals.

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