Developmental validation of a forensic profiling system for black rhinoceros (Diceros bicornis) and extraction of nuclear DNA from historic rhinoceros horns

Title: Developmental validation of a forensic profiling system for black rhinoceros (Diceros bicornis) and extraction of nuclear DNA from historic rhinoceros horns Authors: Dicks, Kara L. Abstract: Demand for rhinoceros horn in the late 20th century led to the population decline of black rhinoceros (Diceros bicornis) by more than 96 %, and the species remains critically endangered. Trade in rhinoceros and their products is prohibited under CITES legislation. Despite this, there has been a recent increase in rhinoceros horn demand for use in traditional Asian medicine (TAM) to treat a range of illnesses including cancer, childhood fevers and as a hangover cure. Consequently, the value of rhinoceros horn in illegal trade has risen dramatically, alongside a dramatic increase in poaching. Large numbers of horns exist in historic collections, and since 2011 there has been numerous thefts in both rhinoceros range states and throughout Europe. Wildlife DNA forensic analysis can be used to individually identify rhinoceros horn, matching a seized horn to a crime scene. This can provide valuable evidence in criminal proceedings. This study therefore aimed to validate a profiling system that could be used for D. bicornis horns and to determine an optimal DNA extraction method for historic horns. A developmental validation of a marker panel consisting of 15 short tandem repeat (STR) and one sexing locus was carried out to determine its reliability and efficacy in singleplex reactions. The markers were not found to deviate from patterns of Mendelian inheritance, and species specificity studies found that only two loci (WR7C and ZF1) amplified human and dog DNA. Alleles could be scored at DNA concentrations of less than 1 ng/μL for all loci except WR7C. The marker panel was found to be highly reproducible with a maximum d-value of 0.545, mean heterozygous balance of 1.30 and stutter ratio of 0.35. The marker panel was also robust to reductions in annealing temperature (TA) and increasing cycle number (up to 43 cycles), although increasing TA reduced amplification success. The markers were then combined into two multiplexes and allele frequency data was generated for 52 D. bicornis michaeli, and two markers (BlRh37D and IR12) were rejected for violating assumptions of Hardy-Weinberg equilibrium and linkage equilibrium. The average probability of identity (PIave) ranged between 1.06 x 10-11 and 1.16 x 10-7 with varying levels of relatedness and population structure. The marker panel was determined suitable for use in forensic casework analysis under the defined conditions, and limitations were described. Previous studies have shown that nuclear DNA (nDNA) can be extracted from modern horns, but how effective those methods would be on historic horns was unknown. Optimisation of a number of aspects of the extraction method was carried out, including sample preparation, chemical breakdown with dithiothreitol (DTT), mass of starting material and extraction kit. Mechanical breakdown of rhinoceros horn material using a mixer mill was determined unnecessary, whilst the addition of DTT was found to improve digestion. Irrespective of extraction kit, 20 mg of horn material yielded sufficient quality and quantity nDNA for forensic profiling. Qiagen extraction kits using the QIAamp Mini columns were found to yield superior DNA quantity compared with QIAamp MinElute columns. Whilst KingFisher Cell and Tissue DNA Kits yielded equivalent extract quantity, mitochondrial DNA contamination between samples was detected. The Qiagen method using QIAamp MinElute columns was therefore considered the most robust extraction method for historic rhinoceros horns. This work describes the conditions and limitations of a forensic profiling system for D. bicornis and an effective extraction methodology for historic rhinoceros horns. This enables this marker panel to be used with confidence in forensic casework analysis of both modern and historic horns.