Abstract

On average, 55,000 deer are illegally killed in the UK each year. Deer which are killed illegally are often done so in a cruel and inhumane way, causing unnecessary distress and suffering to the animal. Furthermore, the illegal killing of deer negatively impacts herd numbers and the natural environment of the deer. Deer are legally killed each year by licenced game keepers in order to keep a control of the ever-growing size of deer herds in the UK. Illegally killing deer disrupts this process and thus affects the livelihood of these workers. Illegally killed deer are often gall roached on site and the sought-after meat and antlers are taken, whilst leaving the carcass to rot where slaughtered. More often than not, the perpetrators get away with their crimes, free to commit again.

During the initial phase of work, five samples were collected and extracted via an optimised method to ensure DNA quantity and quality for whole genome sequencing. The entire genome of the fallow deer was sequenced via Pac Bio Sequencing, using a tissue sample from an individual from Richmond Park, London. This enabled the investigation of the fallow deer genome for STRs within nuclear DNA, in order to design new primers. This overcame issues found with cross-species amplification between fallow deer and primers developed for other species of deer in the UK during a previous project. The Genome is 3,108,385,535 bp in length with the sequence assembled into 35 Chromosomes including an assembly of the X and Y allosomes. The final assembly also had a BUSCO completeness score of 99%. The annotated genome was found to consist of 34,891 genes, 22,157 of which are protein coding genes. The final BUSCO v4.1.4 completeness score for the annotation was 99%. Predicted genes have an average transcript length of 37,945bp and on average each gene has 10 exons.

The second study involved mining the genome for STRs; in order to best assess the entire UK population, 364 samples were collected in total across 15 geographical locations. Out of the 364 samples, the best 190 were used for downstream analysis. Using the genome sequenced in study 1, a total of 21,580 tetra and penta nucleotide STRs were located within the genome and 264 primers were developed. Out of the 132 designed primers for tetranucleotide markers, 90 were tested and 9 were found to be polymorphic. Polymorphism was detected via fragment analysis on the ABI3500 followed by Sanger Sequencing. The lack of polymorphic loci discovered can be attributed to the low genetic diversity of the fallow deer species. It is well documented that Dama dama have low genetic diversity caused by a genetic bottle neck during the Mesolithic (8000 BC – 2700 BC) and Neolithic (10,200 BC - 2000 BC) periods. This lack of genetic diversity which is still observed today implicates the likelihood of a forensically significant STR multiplex for fallow deer individual identification.

Oxford Nanopore Sequencing via the Native Barcoding kit was used to develop a novel method for rapidly screening samples for polymorphism. The results for the NGS rapid polymorphism screening study showed that this new method is an excellent way of quickly screening for genetic variation at known STR loci. This method is quicker and overall more cost effective because more loci can be screened at once against an increased number of samples (223 in this study). Therefore, loci can be easily included or omitted, compared to traditional methods of gel electrophoresis and CE. In terms of polymorphism in fallow deer, the nanopore results showed that there was majorly no genetic variation, however Loci Fallow70, Fallow89, Fallow118, Fallow124 and Fallow129 showed increased variation compared to Sanger Sequencing results.
The mitochondrial genome for Dama dama is 16,322bp in length, consisting of 13 protein coding genes, 2 rRNAs and 22 tRNAs. The notable difference with the mitogenome for Dama dama is that it is the smallest compared to all other Cervidae mitochondrial genomes analysed in this study. Overall, the phylogenetic and divergence time analysis has provided support for previously published theories but has also given a new perspective on the divergence of the Dama dama species and its sub-species Dama mesopotamica. Findings suggest that as Megaloceros giganteus is the older of the Dama species, Dama mesopotamica has held onto more ancestral genetic sequence than Dama dama, and that it is Dama dama that diverged first and has mutated away from the ancestral lineage, around 5.35 million years ago.

This project was the first to utilise the full sequence of the D-Loop to assess population diversity and analyse population signature haplotypes. This study also compared the use of Sanger sequencing to NGS via Oxford Nanopore for the application of SNP and InDel detection. Interestingly, only 6 sites of variation were identified as a result of the sanger sequencing. However, this was latterly increased to 47 sites of variation as a result of the Nanopore sequencing. This increase in variation detected is likely due to the increased sequencing depth achieved with Oxford Nanopore Sequencing. Variation calling via nanopore sequencing was achieved at a 300x sequencing depth compared to 1x with Sanger Sequencing. The level of variation observed in the NGS sequences compared to Sanger equates to a staggering 783% increase.

This project is the first to fully sequence the genome of fallow deer and provides the groundwork for further research involving this species. The result of this project benefits the welfare of the fallow deer species across the world, contributing to the global effort to combat wildlife crime as well as the conservation of the species.