BackgroundWild animals’ meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species.ResultsThe combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as ‘near threatened’; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species.ConclusionsThe reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices.Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.

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CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes.

In this study, the GlobalFiler(®) Express amplification kit was evaluated for forensic use in 541 South African individuals belonging to the Afrikaaner, amaXhosa,(1) amaZulu,(1) Asian Indian and Coloured population groups. Allelic frequencies, genetic diversity parameters and forensic informative metrics were calculated for each population. A total of 301 alleles were observed ranging between 5 and 44.2 repeat units, 43 were rarely observed partial repeats and seven were novel. The combined match probability (CMP) ranged from 2.21×10(-26) (Coloured) to 5.21×10(-25) (AmaZulu), and the combined power of exclusion (CPE) 0.9999999978 (Afrikaaner) to 0.99999999979 (AmaZulu) respectively. No significant departures from Hardy-Weinberg equilibrium (HWE) were observed after Bonferroni correction. Strong evidence of genetic structure was detected using the coancestry coefficient θ, Analysis of Molecular Variance (AMOVA) and an unsupervised Bayesian clustering method (STRUCTURE). The efficiency of assignment of individuals to population groups was evaluated by applying likelihood ratios with WHICHRUN, and the individual ancestral membership probabilities inferred by STRUCTURE. Likelihood ratios performed the best in the assignment of individuals to population groups. Signs of positive selection were detected for TH01 and D13S317 and purifying/balancing selection for locus SE33. These three loci also displayed the largest informativeness for assignment (In) values. The results of this study supports the use of the GlobalFiler(®) STR profiling kit for forensic applications in South Africa with the additional capability to predict ethnicity or continental origin of a random sample.