Ross McEwing

Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing

Caviar‐producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta‐samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired‐end RAD data focused on the identification of SNPs in the paired‐end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population‐wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping‐by‐sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools.

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

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 (theta) and inbreeding (f). Accounting for these estimates, the average probability of identity (PI(ave)) was 2.18 x 10(-7). This case study can act as a framework for others attempting to develop wildlife forensic profiling systems.

Reference‐free SNP discovery for the Eurasian beaver from restriction site–associated DNA paired‐end data

In this study, we used restriction site–associated DNA (RAD) sequencing to discover SNP markers suitable for population genetic and parentage analysis with the aim of using them for monitoring the reintroduction of the Eurasian beaver (Castor fibre) to Scotland. In the absence of a reference genome for beaver, we built contigs and discovered SNPs within them using paired‐end RAD data, so as to have sufficient flanking region around the SNPs to conduct marker design. To do this, we used a simple pipeline which catalogued the Read 1 data in stacks and then used the assembler cortex_var to conduct de novo assembly and genotyping of multiple samples using the Read 2 data. The analysis of around 1.1 billion short reads of sequence data was reduced to a set of 2579 high‐quality candidate SNP markers that were polymorphic in Norwegian and Bavarian beaver. Both laboratory validation of a subset of eight of the SNPs (1.3% error) and internal validation by confirming patterns of Mendelian inheritance in a family group (0.9% error) confirmed the success of this approach.

Genetic data from 28 STR loci for forensic individual identification and parentage analyses in 6 bird of prey species

Twenty-eight STR loci were screened in wild populations of six bird of prey species providing allele frequencies and population genetic parameters necessary for the application of STRs in wildlife forensic genetic casework. Individual STR loci were validated according to forensic recommendations in specimens of golden eagle (Aquila chrysaetos), goshawk (Accipiter gentilis), merlin (Falco columbarius), peregrine falcon (Falco peregrinus), gyr falcon (Falco rusticolus) and saker falcon (Falco cherrug). Deviations from Hardy-Weinberg expectations and linkage disequilibrium between locus pairs were examined. The average probability of identity (PI(ave)) and power of exclusion (PE) suggest the profiling systems of golden eagle, goshawk, merlin and peregrine falcons are capable of providing robust and highly discriminatory forensic evidence for legal proceedings. Due to low sample numbers the allele frequency data for gyr and saker falcons is not currently capable of providing an effective probability of identity. Further work should focus on increasing the size of these data sets.

A DNA-based approach for the forensic identification of Asiatic black bear (Ursus thibetanus) in a traditional Asian medicine.

Abstract:  Attempts to prevent illegal trade in bile and gallbladders from Asiatic black bears, Ursus thibetanus, are hampered by difficulties associated with identifying such items. We extracted DNA from bile crystals of unknown species origin and generated partial cytochrome b (cyt b) sequences using either universal primers (positioned in conserved regions of cyt b), or primers designed on existing U. thibetanus sequences (UT). Species origin was determined by aligning resolved sequences to reference sequence data. The universal primers were unsuitable for U. thibetanus identification when multiple species templates were present in the samples. The UT primers amplified U. thibetanus DNA from all sample extracts, including those containing mixed species templates. The amplified fragment can distinguish U. thibetanus from the most closely related species, U. americanus, a distinct advantage of DNA sequencing over the methods currently used to analyze suspected U. thibetanus bile.

The use of cross-species genome-wide arrays to discover SNP markers for conservation genetics: a case study from Arabian and scimitar-horned oryx

The potential use of single nucleotide polymorphism markers (SNPs) in conservation genetics is widely recognized; however, methods for discovering large numbers of SNPs typically rely on relatively expensive, genome-wide, species-specific research projects which limits their development in many taxa. Here we describe the use of high-density SNP genotyping arrays designed for cattle to discover SNPs in two antelope species. From a total of 54,001 SNP markers on the array, the analysis yielded 148 polymorphic markers in the scimitar-horned oryx and 149 in the Arabian oryx. The results represent a first step toward developing SNP marker panels for ongoing projects on each species. As high density genotyping arrays become available for an increasing number of model species, this approach has the potential to generate SNP markers, rapidly and affordably, in a broad range of species for conservation genetic research.

Genetic data from 15 STR loci for forensic individual identification and parentage analyses in UK domestic dogs (Canis lupus familiaris)

Eighteen STR loci and one sex determination locus present in the Finnzymes Canine 2.1 STR Multiplex Reagent Kit were screened in the UK dog population providing allele frequencies and population genetic parameters necessary for the application of STRs to forensic genetic casework. A total of 375 dogs were genotyped, including representative samples from each of twelve breeds used to evaluate Hardy-Weinberg equilibrium and calculate inter-population pairwise F(ST) values. Three loci were excluded from calculations of average random match probability due to deviations from Hardy-Weinberg Equilibrium or ambiguous genotyping. Random match probability based on fifteen STR loci and one sex locus was subsequently estimated to be 2.8 × 10(-17) for unrelated individuals across breeds.

Preliminary data suggests genetic distinctiveness of gyr and saker falcons

The inadequately controlled trade in gyr and saker falcons has lead to saker falcons becoming endangered and both species being CITES listed. However, the phylogenetic relationship between these nominal species is unresolved preventing their molecular identification and limiting the availability of data for conservation management. This study amplified DNA from the mitochondrial cytochrome oxidase I (COI) gene and nine nuclear microsatellite markers to highlight previously unobserved genetic differences between these two putative species. Results show that gyr and saker are paraphyletic using COI and therefore indistinguishable using this marker. However, the microsatellite allele frequency differences suggest that it is possible to assign an unidentified bird to its correct species with 98% accuracy. The results suggest the two species should be monitored separately to ensure short term conservation success.

Genetic assessment of the Arabian oryx founder population in the Emirate of Abu Dhabi, UAE: an example of evaluating unmanaged captive stocks for reintroduction

Since being declared extinct in the wild in 1972, the Arabian oryx has been the subject of intense and sustained effort to maintain a healthy captive population and to reintroduce the species to its ancestral range. Previous reintroductions and associated genetic assessments focused on the release of closely managed zoo animals into Oman and included observations of inbreeding and outbreeding depression. Here we describe the use of multiple unmanaged herds as source populations for a new reintroduction project in the United Arab Emirates, allowing a comparison between studbook management and uncontrolled semi-captive breeding approaches to the conservation of genetic diversity. Results of mitochondrial control region sequencing and 13-locus microsatellite profiling highlight a severe lack of diversity within individual source populations, but a level of differentiation among populations that supports the formation of a mixed founder herd. The combined release group contained a similar level of diversity to each of the intensively managed captive populations. The research includes the first genetic data for animals held on Sir Bani Yas Island, a former private reserve which until recently held over 50% of the world’s Arabian and scimitar-horned oryx and is recognized as having huge potential for re-establishing endangered antelope species in the wild. The genetic assessment provides the first stage of an ongoing genetic monitoring programme to support future supplemental releases, translocations and genetic management of reintroduced populations.

An allelic discrimination SNP assay for distinguishing the mitochondrial lineages of European wildcats and domestic cats

Here we present an allelic discrimination assay designed to distinguish European wildcat mtDNA lineages from those of the domestic cat. Introgression between the native wildcat and introduced domestic cat has the potential to limit and reduce the recent recovery of remnant populations. Applied conservation genetic techniques can aid current conservation decisions on lethal control and neutering measures in hybrid zones. This real-time PCR technique offers a rapid, inexpensive and reliable assay to assess mtDNA introgression in the wild and has already identified hybrid individuals in the Scottish wildcat captive breeding programme.