Deep sequencing approaches to Dugong (Dugong dugon) population genetics and diet

Elizabeth Ann Baker

The role of conservation genetics is, with the aid of DNA markers, to characterise the genetic health of populations with the aim to preserve or restore levels of genetic diversity within a species across its range. The use of ancient or historical DNA approaches to conservation genetic studies adds a temporal dimension, providing a reference for current levels of genetic diversity to that in the past. The complicating factors associated with degraded DNA are the facts that DNA template can be fragmented, damaged and difficult to characterise. Next generation sequencing (NGS) platforms are able to overcome some of the issues associated with degraded DNA and therefore an NGS approach is used in this thesis to explore the historical bone and faecal material from the endangered Dugong (Dugong dugon). The dugong's Australian range covers the northern shallow coastal waters, extending from Shark Bay in Western Australia, to Moreton Bay on the east coast of Queensland with two distinct maternal lineages identified through the mitochondrial control region (Blair et al., in press). Deep sequencing of 322bp of mitochondrial control region amplicons from 150 historical bone samples (30- 50 years old) yielded 100 (67%) high-fidelity sequences. The results herein describe 11 previously uncharacterised haplotypes. Importantly the bone samples studied provide additional samples from key locations that were not well represented in the modern study of Blair et al. (in press). In a pilot study, deep sequencing also facilitated the generation of microsatellite data for nine historical dugong samples. By directly sequencing genotypes for the locus DduB02 for the nine samples alleles could be identified and single nucleotide polymorphism were also identified. Once optimised this approach holds great promise for the accurate sequencing of microsatellites especially on degraded DNA. DNA preservation was also investigated in seven dugong faecal samples with the aim to see if this non-invasive substrate is suitable for genetic analyses. Dugong mtDNA was amplified from each of the samples and high throughput sequencing of the trnL gene identified the floral components. The pilot dietary study demonstrates the potential of the approach but highlighted that, at this point in time; a lack of coverage of seagrass trnL sequence on GenBank was a limiting factor. The varied applications of next generation deep sequencing showcased in this thesis have highlighted potential avenues for ongoing research such as historical and modern phylogeography and population genetics studies using challenging substrates, further insights into the biology and feeding ecology of dugongs, and novel methodological approaches and techniques that can be applied across a wide range of taxa, especially those of conservation concern.

Deep sequencing approaches to Dugong (Dugong dugon) population genetics and diet

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