Classified as critically endangered since 1994, the Trinidad Piping guan (Pipile pipile) is an endemic species estimated to number less than 200 individuals. Known to locals of Trinidad as the ‘Pawi’ this bird has been the subject of substantial hunting pressures and much of the species habitat has been destroyed through deforestation. Although officially protected since 1958, occasional recreational hunting of this elusive species still occurs. Due to difficulties locating and capturing the species, no genetic research has previously been performed using samples obtained from Trinidad. All previous research studies have been conducted using biological materials obtained from captive birds outside Trinidad and island data has never been obtained or compared. The genetic diversity of the remaining population was therefore examined through the investigation of mitochondrial haplotypes, pairwise comparison and SNP analysis. With the intention of assisting the protection of this endangered species by the location of remaining areas of habitation, methods of genetic identification were established for the Trinidad Piping guan utilising non-invasive feather samples. Species specific primers were created in the regions of the ND2 and cyt b genes of the mitochondrial genome to identify Pipile pipile. Species detection was further verified with the use of PCR-RFLP of the same gene regions digested with BsaXI, EcoRV and BsrDI. This combined approach allowed the separation of closely related taxa based on single inter-species SNPs. Confirmation of species identification was subsequently performed through the use of forensically informative nucleotide sequencing. The established methodologies were used in the current study to correct the classification of a UK breeding population of Piping guans thought to be Pipile pipile and to identify Trinidad field samples. These detection methods have implications for ecological studies through the location of populations from trace evidence collected in the field. In addition this method could be used to assist Trinidadian police forces in the identification of bushmeats or simply act as a deterrent to hunters. The sequence data obtained in the present study were also used to re-assess the phylogeny of Piping guans. As genetic sequence from a true island bird was previously unstudied, differences between phylogenies created using non-island and island bird data sets were examined. Combined analysis was performed on 1884bp of the ND2 and cyt b genes and placement of Trinidad Piping guan was found to differ from that which has been previously published.

<p>A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.</p> <p>I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.</p> <p>To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.</p> / Dissertation