A forensic STR profiling system for the Eurasian badger: A framework for developing profiling systems for wildlife species. Forensic Sci

Abstract Developing short tandem repeat (STR) profiling systems for forensic identification is complicated in animal species. Obtaining a representative number of individuals from populations, limited access to family groups and a lack of developed STR markers can make adhering to human forensic guidelines difficult. Furthermore, a lack of animal specific guidelines may explain why many wildlife forensic STR profiling systems developed to date have not appropriately addressed areas such as marker validation or the publication and analysis of population data necessary for the application of these tools to forensic science. Here we present a methodology used to develop an STR profiling system for a legally protected wildlife species, the Eurasian badger Meles meles. Ten previously isolated STR loci were selected based on their level of polymorphism, adherence to Hardy-Weinberg expectations and their fragment size. Each locus was individually validated with respect to its reproducibility, inheritance, species specificity, DNA template concentration and thermocycling parameters. The effects of chemical, substrate and environmental exposure were also investigated. All ten STR loci provided reliable and reproducible results, and optimal amplification conditions were defined. Allele frequencies from 20 representative populations in England and Wales are presented and used to calculate the level of population substructure (u) and inbreeding ( f). Accounting for these estimates, the average probability of identity (PI ave ) was 2.18 Â 10 À7 . This case study can act as a framework for others attempting to develop wildlife forensic profiling systems.

Phylogeography of the Sunda pangolin, Manis javanica: Implications for taxonomy, conservation management and wildlife forensics

The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD‐seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the ‘out of Borneo hypothesis’, whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range‐wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios

Distribution of polymorphic, monomorphic and failed SNPs mapped to the dog (<b><i>Canis lupus familiaris</i></b><b>) genome (shown as three rows, </b><b><i>n</i></b><b> = 193, </b><b><i>n</i></b><b> = 33,131 and </b><b><i>n</i></b><b> = 136,903 respectively).</b>

<p>Distribution of polymorphic, monomorphic and failed SNPs mapped to the dog (<b><i>Canis lupus familiaris</i></b><b>) genome (shown as three rows, </b><b><i>n</i></b><b> = 193, </b><b><i>n</i></b><b> = 33,131 and </b><b><i>n</i></b><b> = 136,903 respectively).</b></p

Example genotypes obtained for locus BICF2G630131208 using KASP chemistry and an ABI Step-One real-time PCR machine.

<p>The three discrete clusters of heterozygous and alternative homozygous genotypes denoted by green, red and blue points respectively include all 24 Antarctic fur seals as well as positive canine control samples. The two black squares indicate negative controls.</p

Distribution of the distance between each SNPs and its nearest gene in the dog, shown for polymorphic, monomorphic and failed SNPs.

<p>Distribution of the distance between each SNPs and its nearest gene in the dog, shown for polymorphic, monomorphic and failed SNPs.</p

Examples of single nucleotide polymorphisms (SNPs) from the canine SNP chip that cross-amplify in Antarctic fur seals.

<p>Each point represents a single sample. ‘Norm R’ (y-axis) is the normalized sum of the intensities of the two channels (Cy3 and Cy5). ‘Norm Theta’ (x-axis) is ((2 / p)Tan)1 (Cy5 / Cy3)) where a value near 0 represents a homozygote for allele A (denoted by red points) and a value near 1 represents a homozygote for allele B (denoted by blue points). Heterozygotes fall approximately mid-way between these values and are denoted by purple points. The numbers of samples called by GenomeStudio for each of the three possible genotypes are shown below the x-axis. (a–d) Classical three-cluster patterns for SNPs considered successful and polymorphic; (e) A monomorphic SNP; (f) A locus that failed to yield an interpretable assay and was thus classified as a genotyping failure.</p

Details of seven polymorphic SNPs revealing homology to the <i>Arctocephalus gazella</i> transcriptome.

<p>Details of seven polymorphic SNPs revealing homology to the <i>Arctocephalus gazella</i> transcriptome.</p

Molecular sexing of African rhinoceros

We report the development of a fast and reliable PCR-based method for sex identification of African Rhinoceros (Ceratotherium simum and Diceros bicornis) that could easily be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. In both species, the amplified ZFX and ZFY amplicons differ in size by 7 bp and can thus be differentiated by capillary electrophoresis. Blood, tissue, horn, and faecal samples were correctly sexed using this method. Cross species testing also demonstrated that this method could be used to sex Indian rhinoceros (Rhinoceros unicornis) samples.Objective 1—European Social Fund