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Exonic Trinucleotide Microsatellites: Applying Genomic and Bioinformatic Techniques to Wildlife Forensic Science

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Exonic Trinucleotide Microsatellites: Applying Genomic and Bioinformatic Techniques to Wildlife Forensic Science Exonic Trinucleotide Microsatellites: Applying Genomic and Bioinformatic Techniques to Wildlife Forensic Science A Thesis Submitted to the Committee on Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Faculty of Arts and Science Amanda M. MacDonald TRENT UNIVERSITY Peterborough, Ontario, Canada © Copyright by Amanda M. MacDonald, 2015 Environmental and Life Science M.Sc. Graduate Program May 2015 ABSTRACT Exonic Trinucleotide Microsatellites: Applying Genomic and Bioinformatic Techniques to Wildlife Forensic Science Amanda M. MacDonald Trinucleotide repeats (TNRs) are a class of highly polymorphic microsatellites which occur in neutral and non-neutral loci and may provide utility for individual- and population- identification. Exonic trinucleotide motifs, in particular, offer additional advantages for non-human species that typically utilize dinucleotide microsatellite loci. Specifically, the reduction of technical artifacts, greater separation of alleles and greater specificity of amplification products leading to more efficient multiplexing and cross-taxa utilization. This study aims to identify and characterize polymorphic trinucleotide repeats and conserved primer sequences which are conserved across Cervidae (deer) species and their potential for individual identification in forensic wildlife investigations. Chapter one provides a broad introduction to trinucleotide microsatellites, chapter two deals with data- mining TNRs and chapter three applies the identified TNRs as genetic markers for individual identification. Results demonstrate proof-of-concept that exonic TNRs are capable of giving random match probabilities low enough to be employed in individual identification of evidentiary samples. Keywords: exons, trinucleotides, wildlife forensics, genetic markers, individual- identification, population-identification, non-human forensics ii ACKNOWLEDGEMENTS Rose & Bernie MacDonald David Leland Paul Wilson Barry Saville Christopher Kyle Candace Gainer Peter MacDonald Mike Illes Rhonda Smith Guy Colton Anna Cappelli Jenifer Richardson Jane Rennie Linda Cardwell iii Table of Contents List of Figures .................................................................................................................................. vi List of Tables .................................................................................................................................. vii Chapter 1: Exonic Trinucleotide Microsatellites and Applications to Wildlife Forensic Science ..... 1 1. Introduction ............................................................................................................................. 1 1.1 Role of trinucleotide repeats in human disease ................................................................ 2 1.2 Trinucleotide repeats and natural functionality in animals ............................................... 6 1.3 Population and geographic patterns of variability............................................................. 7 1.4 Potential applications ........................................................................................................ 8 2. Wildlife Forensic Science........................................................................................................ 10 2.1 Individual Identification ................................................................................................... 11 2.2 Population Identification ................................................................................................. 11 2.3 Trinucleotides in Practice ................................................................................................. 12 3. Future Investment into Trinucleotide Research .................................................................... 13 4. Conclusion .............................................................................................................................. 14 Chapter 2: Establishing search criteria for highly polymorphic exonic trinucleotide motifs ......... 15 1. Introduction ........................................................................................................................... 15 1.1 Nomenclature .................................................................................................................. 15 1.2 Searching for Repeats ...................................................................................................... 17 1.3 Transferability across Species .......................................................................................... 19 2. Methods ................................................................................................................................. 20 2.1 Searching for Exonic Trinucleotide Repeats in the Bovine Genome ................................ 20 3. Results .................................................................................................................................... 23 4. Discussion & Recommendations ............................................................................................ 24 Chapter 3: Exonic Trinucleotide Microsatellites for Individual Identification in Wildlife Forensic Science. .......................................................................................................................................... 27 1. Introduction ........................................................................................................................... 27 2. Materials and Methods .......................................................................................................... 29 2.1 Sample Selection .............................................................................................................. 29 2.2 Sample Preparation .......................................................................................................... 29 2.3 Genotyping ....................................................................................................................... 29 2.4 Statistical Analysis ............................................................................................................ 30 3. Results .................................................................................................................................... 31 3.1 Optimization & Genotyping ............................................................................................. 31 iv 3.2 Levels of Polymorphism ................................................................................................... 32 3.3 Statistical Analysis ............................................................................................................ 38 4. Discussion & Conclusion ........................................................................................................ 43 References ..................................................................................................................................... 48 Appendix 1: Loci data-mined from polyamino-acid search in Genome Browser. ......................... 58 Appendix 2: Primer Sequences Tested for Polymorphism ............................................................ 66 Appendix 3: Electropherograms for loci involved in large animal screen (moose). ...................... 70 Electropherograms for loci involved in large animal screen (caribou). ......................................... 77 Appendix 4: Successfully amplified loci across species including fluorescent dye and optimal annealing temperature. ................................................................................................................. 84 Appendix 5: Allele count and frequencies for polymorphic loci tested in the large animal screening of moose for 5 populations, generated using GenAlEx 6.5. .......................................... 86 Allele count (A.C) and frequencies (F) for polymorphic loci tested in the large animal screening of caribou for 7 populations, generated using GenAlEx 6.5. ............................................................. 88 Appendix 6: PIsibs for moose loci found to be within Hardy-Weinberg Equilibrium. ................... 91 Cumulative PIsibs by locus for moose loci found to be within Hardy-Weinberg Equilibrium. The number corresponds to the numbered loci in the table for PIsibs (moose). ................................ 91 PIsibs for caribou loci found to be within Hardy-Weinberg Equilibrium. ...................................... 92 Cumulative PIsibs by locus for caribou loci found to be within Hardy-Weinberg Equilibrium. The number corresponds to the numbered loci in the table for PIsibs (caribou). ............................... 92 v List of Figures Figure 1 QIAxcel image of 11 individual caribou amplicons at FAM193 locus at initial screening stage showing evidence of monomorphism. .................................................................................................. 34 Figure 2 QIAxcel image of 11 individual caribou amplicons at SLC24A1 locus at initial screening stage showing evidence of polymorphism. .................................................................................................... 34 Figure 3 Electropherogram showing allele morphology of Pen Island caribou samples at SHE locus. 36 Figure 4 Electropherogram showing allele morphology of NWT moose samples at FOXP2 locus. .... 36 vi List of Tables Table 1 Parameter settings used while searching for exonic trinucleotide repeats in the UCSC Table Browser
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