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Sharealike 4.0 International License. How to Cite This This work is licensed under a Creative Commons Attribution-NonCommercial- ShareAlike 4.0 International License. How to cite this thesis / dissertation (APA referencing method): Surname, Initial(s). (Date). Title of doctoral thesis (Doctoral thesis). Retrieved from http://scholar.ufs.ac.za/rest of thesis URL on KovsieScholar Surname, Initial(s). (Date). Title of master’s dissertation (Master’s dissertation). Retrieved from http://scholar.ufs.ac.za/rest of thesis URL on KovsieScholar Genetic diversity and performance trait analysis of the SA Boerperd N. Breytenbach Dissertation submitted in fulfilment of the requirements for the degree of Magister Scientiae in the Faculty of Natural and Agricultural Sciences, Department of Genetics, University of the Free State. Supervisor: H. Bindeman Co-supervisor: Prof. J.P. Grobler 2018 Genetic diversity and performance trait analysis of the SA Boerperd (SA Boerperd Breeders Society) Nadia Breytenbach Department of Genetics University of the Free State 2018 DECLARATION I, Nadia Breytenbach, declare that the Master’s Degree research dissertation that I herewith submit for the Master’s Degree qualification in Genetics at the University of the Free State is my independent work, and that I have not previously submitted it for a qualification at another institution of higher education. _________________ Nadia Breytenbach 2018 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to everyone who made the completion of this dissertation a reality: My supervisors, Mrs. H. Bindeman and Prof J.P. Grobler, for your support and guidance not only throughout the duration of this study, but during my entire academic career. Thank you for allowing me to pursue my passion for horses. The personnel and students of the Genetics Department for their continual encouragement during my project. The SA Boerperd Breeders Society for providing support and necessary information about the SA Boerperd. Special thanks to Mrs. E. Prinsloo for her continual assistance and patience, as well as Dr P. Grové for her enthusiastic conversations and aid in obtaining hair samples. All the breeders and owners who allowed me to sample their horses. Thank you for also sharing your insights and knowledge about the breed. Unistel Medical Laboratories (Dr. Marx and H. Theron) for providing the STR marker data of the registered horses. L.H.P. van de Goor (Dr. Van Haeringen Laboratorium, The Netherlands) and Dr K. Ehlers (University of the Free State) permitting the use of their equine DNA data published in previous works. Lastly, Laetitia and Naas Breytenbach, thank you for always believing in me. To the rest of my family and friends, thank you for your continual support and interest in my project. THANK YOU! Table of contents LIST OF ABBREVIATIONS AND SYMBOLS i LIST OF FIGURES v LIST OF TABLES vi CHAPTER 1: INTRODUCTION TO THE EQUINE INDUSTRY 1. Introduction 1 1.1. Equine breeding 1 1.1.1. Traits under selection 2 1.1.2. Breed studbooks 4 1.2. The South African Boerperd 5 1.2.1. Breed history 5 1.2.2. Breed standard 8 1.2.3. Importance of the SA Boerperd breed 9 1.3. Equine performance 10 1.3.1. Performance traits in horses 11 1.3.1.1. Height in horses 11 1.3.1.2. Gait in horses 12 1.3.2. Genetic analysis of performance traits 13 1.3.2.1. Genetics of height in horses 15 1.3.2.2. Genetics of gaitedness in horses 17 1.3.3. SA Boerperd trait investigations 19 1.4. Equine genetic diversity 19 1.4.1. Factors influencing loss of diversity 20 1.4.2. Measuring equine genetic diversity 21 1.4.2.1. Microsatellite markers 22 1.4.2.2. Single nucleotide polymorphisms 23 1.4.3. Genetic diversity of the SA Boerperd 24 1.5. Aims of the current study 26 References 27 CHAPTER 2: PERFORMANCE ANALYSIS OF THE SA BOERPERD 2.1. Introduction 38 2.2. Materials and methods 40 2.2.1. Ethics statement 40 2.2.2. Sample collection 40 2.2.3. SNP markers used 41 2.2.4. Molecular techniques 42 2.2.5. Data analysis 44 2.3. Results 44 2.4. Discussion and conclusion 49 2.4.1. Height at withers 49 2.4.2. Gaitedness 52 References 55 CHAPTER 3: GENETIC DIVERSITY OF THE SA BOERPERD 3.1 Introduction 59 3.2. Materials and methods 60 3.2.1. Data collection 60 3.2.2. Statistical analysis 62 3.2.2.1. Data organization 62 3.2.2.2. Genetic diversity 63 3.2.2.3. Hardy-Weinberg Equilibrium 63 3.2.2.4. Genetic differentiation and drift 64 3.2.2.5. Individual assignment 64 3.3. Results 65 3.3.1. Within-breed analysis of the SA Boerperd 65 3.3.2. Among-breed analysis 70 3.4. Discussion and conclusion 78 3.4.1. Within-breed diversity and differentiation 78 3.4.2. Differences between breeds 79 References 81 CHAPTER 4: GENERAL DISCUSSION, CONCLUSION & RECOMMENDATIONS 4.1. Performance trait analysis 85 4.2. Genetic diversity 86 4.3. Recommendations 87 References 89 SUMMARY 91 APPENDIX A: SA Boerperd DNA sequence data List of abbreviations and symbols Symbols Meaning °C Degrees Celsius % Percentage ΔK DeltaK ® Registered trademark ™ Trademark Abbreviation Meaning μl Microliter A Adenine ABI Applied Biosystems a.k.a. Also known as AND Andalusian AP-1 Activator protein-1 transcription factor complex APP Appaloosa ARA Arabian BIEC2 Broad Institute Equus caballus version 2.0 BLUP Best linear unbiased prediction bp Base-pair(s) C Cytosine CBP Cape Boerperd i cm Centimetre DMRT3 Double-sex and mab-3-related transcription factor 3 gene DNA Deoxyribonucleic acid EBVs Estimated breeding values ECA Equus caballus autosome / horse chromosome et al. et alli: and others f Frequency F Inbreeding coefficient F Female FEI Fédération Equestre Internationale FIS Population inbreeding coefficient FIT Global inbreeding coefficient FRI Friesian FST Genetic differentiation G Guanine g Gravitational force GBVs Genomic estimated breeding values H2O Water HAC Hackney He Expected heterozygosity hh Hands high Ho Observed heterozygosity HMGA2 High mobility group AT-hook 2 gene ii HWE Hardy-Weinberg equilibrium HZ Unbiased heterozygosity ICE Icelandic Horse ISAG International Society for Animal Genetics K Number of clusters kb Kilo base-pair(s) kg kilogram LASP1 LIM and SH3 protein 1 gene LCORL Ligand dependent nuclear receptor corepressor-like gene LD Linkage disequilibrium M Male MCMC Markov Chain Monte Carlo mg Milligram MgCl2 Magnesium chloride ml Millilitre mm Millimetre MSTN Myostatin mRNA Mitochondrial ribonucleic acid mtDNA Mitochondrial deoxyribonucleic acid n Number of chromosomes N Sample size Na Mean number of alleles NA Not applicable iii Ne Effective number of alleles PA Private alleles PCR Polymerase chain reaction PF Forward primer PR Reverse primer QTL Quantitative trait loci rpm Revolutions per minute SA South African SAB South African Boerperd SD Standard deviation SNPs Single nucleotide polymorphisms ST Stud STA Standardbred STRs Short tandem repeat markers T Thymine TEN Tennessee Walker TFIID Transcription factor IID gene THO Thoroughbred UFS University of the Free State UML Unistel Medical Laboratories v Version ZFAT Zinc finger and AT hook domain containing gene iv List of figures Figure Title Page Figure 1.1 Size difference between a Shire and American Miniature. 3 Figure 1.2 Icelandic Horse displaying the ambling gait, tölt. 3 Figure 1.3 Transvaal Burgher astride his Boerperd during the Anglo Boer War. 6 Figure 1.4 Example of a SA Boerperd. 8 Figure 1.5 SA Boerperd brand marking. 8 Figure 1.6 Position for measuring equine height at withers. 11 Figure 1.7 SA Boerperd exhibiting the rack gait. 13 Figure 2.1 South African map indicating sampling locations of SA Boerperd 41 individuals. Figure 3.1 South African map indicating the 12 stud localities from which 61 microsatellite data were obtained. Figure 3.2 Graphical representation of the population structure of 363 69 SA Boerperd horses from 12 studs (K=2). Figure 3.3 Plot indicating the number of clusters (K) that best fit the genomic 69 data of 12 SA Boerperd studs. Figure 3.4 Plot indicating the mean likelihood L(K) and variance per K-value for 70 12 SA Boerperd studs obtained from STRUCTURE. Figure 3.5 Number of clusters (K) that best fit the genomic data of 10 equine 75 breeds. Figure 3.6 Mean likelihood L(K) and variance per K-value for 10 equine breeds. 76 Figure 3.7 Clustering output for three values of K in 3,275 horses belonging to 10 76 breeds. v List of tables Table Title Page Table 1.1 Estimated genotype frequencies for the DMRT3_Ser301STOP 18 mutation (C>A) and the closely linked SNP BIEC2_620109 (C>T) in a total of 2,749 horses across different breeds. Table 1.2 Estimates of genetic variability for South African and selected 24 other domestic horse breeds. Table 1.3 Genetic variability at 12 loci in seven horse populations. 25 Table 2.1 Investigated SNPs, their chromosomal positions, primer 42 sequences, annealing temperatures and references. Table 2.2 Gender, height, gait-ability and diversity at performance-related 45 SNPs in 100 SA Boerperd horses. Table 2.3 Average height (cm) of each size-associated genotype within the 47 SA Boerperd. Table 2.4 Proportions of 3/5-gaited individuals for the SNP 47 DMRT3_Ser301STOP (C > A). Table 2.5 Contingency table input of the SNP data used to perform the 48 Fisher’s exact test. Table 2.6 Distributions of the genotype and allelic frequencies of the four 48 investigated SNPs in the SA Boerperd. Table 3.1 The conversion of ISAG (alphabetical) nomenclature to the number 62 of repeat nomenclature. Table 3.2 Genetic diversity of 12 SA Boerperd populations based on 17 66 microsatellite markers.
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