Reliable and robust molecular sexing of the hen harrier (Circus cyaneus) using PCR-RFLP of the CHD1 gene

The hen harrier (Circus cyaneus) is a bird of prey that is persecuted in the United Kingdom, and there is a need for a DNA-based individual identification and sexing system for the use in forensic investigations. This study reports a new set of PCR primers for the chromo-helicase-DNA-binding protein 1 gene, which allows sexing using PCR-RFLP. Instead of exonic primers that amplify across a large intron, this set consists of a primer within the intron, enabling reduction in amplicon sizes from 356 to 212 bp and 565 to 219 bp in W and Z chromosomes. DNA degradation and dilution experiments demonstrate that this set is significantly more robust than one that amplifies across the intron, and sequencing of the intronic primer-binding region across several individuals shows that it is highly conserved. While our objective is to incorporate this primer set into an STR-based individualization kit, it may in the meantime prove useful in forensic or conservation studies

Metabarcoding-based dietary analysis of hen harrier (Circus cyaneus) in Great Britain using buccal swabs from chicks

The hen harrier is a heavily persecuted bird of prey in Great Britain since its diet includes Red grouse, a game bird shot in driven and walked-up grouse shooting. Unlike walked-up shooting where shooters walk up moors and flush grouse with dogs, in driven grouse shooting the grouse are driven by beaters towards static shooters. Driven grouse moors (DGMs) are increasingly being managed to sustain high densities of Red grouse intensifying a long-standing conservation conflict between conservationists and grouse moor keepers. A metabarcoding approach was used with degenerate universal cytochrome oxidase I and cytochrome b primers along with hen harrier blocking primers. A novel sampling method was used to detect prey in buccal swab samples from chicks from broods across Great Britain from both managed and unmanaged moorland habitats. This resulted in detection of 62 species of prey across 51 broods with Meadow pipit, Red grouse, Wren, Skylark, and voles being most frequently detected. Frequency of occurrence data and species accumulation curves reveal high incidence of Red grouse and low prey species richness in the diet of hen harriers in DGMs but low incidence of Red grouse and high prey species richness in walked-up and unmanaged moors. Waders were only detected within walked-up and unmanaged moors and not within DGMs where they have been reported to occur at high densities. Regional species detected included endemic species such as the Orkney vole seen only in Orkney. This study represents the first metabarcoding-based dietary analysis in a raptor using buccal swabs

SkydancerPlex: A novel STR multiplex validated for forensic use in the hen harrier (Circus cyaneus)

The hen harrier (Circus cyaneus) is a bird of prey which is heavily persecuted in the UK because it preys on the game bird red grouse (Lagopus lagopus scoticus). To help investigations into illegal killings of hen harrier, a STR multiplex kit containing eight short tandem repeat (STR) markers and a chromohelicase DNA binding protein 1 (CHD 1) sexing marker was developed. The multiplex kit was tested for species specificity, sensitivity, robustness, precision, accuracy and stability. Full profiles were obtained with as little as 0.25 ng of template DNA. Concurrent development of an allelic ladder to ensure reliable and accurate allele designation across laboratories makes the SkydancerPlex the first forensic DNA profiling system in a species of wildlife to be fully validated according to SWGDAM and ISFG recommendations. An average profile frequency of 3.67 × 10(-8), a PID estimate of 5.3 × 10(-9) and a PID-SIB estimate of 9.7 × 10(-4) make the SkydancerPlex an extremely powerful kit for individualisation

Fate of novel DNA in transgenic rainbow trout (Oncorhynchus mykiss)

Various aspects concerning the fate of novel DNA in rainbow trout were investigated. These included mosaicism, germ-line transmission, and the methylation status of a mouse metallothionein-1/rat growth hormone gene (mMTrGH) construct, in addition to the fate and temporal expression patterns of injected DNA sequences during development, using a carp β-actin/CAT gene (pFV1) construct. Widespread mosaicism was observed amongst the transgenic fish, resulting in an unequal distribution of transgene copies in the cells/tissues of the fish. Germ line transmission was, however, observed in 50&#37; of the transgenic fish containing the mMTrGH gene construct, with transmission frequencies ranging from 14-81&#37;. The lack of transmission from the rest of the founder transgenics could also be attributed to mosaicism. The methylation studies revealed tissue-specific differences in the methylation patterns of the mMTrGH transgene, but could not be correlated with expression due to the consistent lack of expression of this transgene in our founder transgenics. With respect to the fate of DNA during development, prolonged persistence of injected DNA sequences appeared likely, probably contributing to the mosaicism observed. Studies on the conformational influences of injected DNA revealed that linear molecules were more efficient at forming concatemers and persisting through development. They, therefore, resulted in high levels of expression, and perhaps greater rates of integration. The concatemers generated with circular injected molecules appeared to consist of head to tail arrangements of transgene copies, whereas those generated with linear injected molecules appeared to consist of random arrangements of transgene copies.</p

Forensic DNA analysis for animal protection and biodiversity conservation: A review

The use of DNA analysis in forensic investigations into animal persecution and biodiversity conservation is now commonplace and crimes such as illegal collection/smuggling, poaching, and illegal trade of protected species are increasingly being investigated using DNA based evidence in many countries. Using DNA analysis, it is possible to identify the species and geographical origin (i.e. population) of a forensic sample, and to also individualise the sample with high levels of probability. Despite extensive literature in animal species, there is unfortunately a serious lack of information on plant species, with only a handful of recent studies. In this review, I detail the applications and diverse forensic investigations that have been carried out to date whilst also highlighting recent developmental studies which offer forensic potential for many species in the future

Use of non-human DNA analysis in forensic science: a mini review

Analysis of non-human DNA in forensic science, first reported about two decades ago, is now commonplace. Results have been used as evidence in court in a variety of cases ranging from abduction and murder to patent infringement and dog attack. DNA from diverse species, including commonly encountered pets such as dogs and cats, to plants, viruses and bacteria has been used and the sheer potential offered by such analyses has been proven. In this review, using case examples throughout, we detail the considerable literature in this field

Phylogenetic Evidence for a Case of Misleading Rather than Mislabeling in Caviar in the United Kingdom

Sturgeons and paddlefish are freshwater fish which are highly valued for their caviar. Despite the fact that every single species of sturgeon and paddlefish is listed under CITES, there are reports of illegal trade in caviar where products are deliberately mislabeled. Three samples of caviar purchased in the United Kingdom were investigated for accurate CITES labeling using COI and cyt b sequencing. Initial species identification was carried out using BLAST followed by phylogenetic analyses using both maximum parsimony and maximum likelihood methods. Results showed no evidence for mislabeling with respect to CITES labels in any of the three samples, but we observed clear evidence for a case of misleading the customer in one sample

Regulation and expression of transgenes in fish - A review

Transgenic fish, owing to a number of advantages which they offer over other species, are proving to be valuable model systems for the study of gene regulation and developmental genetics in addition to being useful targets for the genetic manipulation of commercially important traits. Despite having begun only a decade ago. the production of transgenic fish has become commonplace in a number of laboratories world-wide and considerable progress has been made. In this review, we initially consider the various regulatory elements and coding genes which have been used in fish, and subsequently discuss and compare both the transient and long-term fate and expression patterns of injected DNA sequences in the context of the different factors which are likely to have an effect on the expression of transgenes.</p