CROSS-SPECIES MICROSATELLITE MARKERS FOR THE DETECTION OF HYBRIDS IN THE GENUS CONNOCHAETES Letecia Wessels Dissertation submitted in accordance with 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: Prof. J.P. Grobler Co-supervisors: Prof. A. Kotze Dr. K. Ehlers July 2013 DECLARATION DECLARATION I certify that the dissertation hereby submitted for the degree M.Sc. Genetics at the University of the Free State is my independent effort and had not previously been submitted for a degree at another University/Faculty. I furthermore waive copyright of the dissertation in favour of the University of the Free State ____________________________ L. Wessels ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would like to express my sincere gratitude to the following people who made it possible for me to complete my dissertation: My supervisors, Prof. J.P. Grobler, Prof. A. Kotze and Dr. K. Ehlers, for your precious time, patience and guidance throughout the duration of this study. The National Research Foundation (NRF) and the National Zoological Gardens (NZG) of South Africa for funding this project. A very special word of thanks to Mr. S. Vrahimis, Mr. J. Watson and other staff members of the Department of Economic, Tourism and Environmental Affairs (DETEA) for collecting the samples for this project, without your commitment this project would not have been possible. To Mr. S. Vrahimis I would like to also express my gratitude for providing me with your expert knowledge regarding wildebeest in South Africa. I would like to thank all the staff members at the Department of Genetics, University of the Free State, for your continued support. To Prof. J.J. Spies for always showing interest in my project and allowing me the time to complete my dissertation. Then last but not least, my parents Chris and Lynette Jonker and my husband Albert Wessels, thank you for always believing in me and supporting me in all aspects of life. TABLE OF CONTENTS TABLE OF CONTENTS Page LIST OF ABBREVIATIONS AND SYMBOLS i LIST OF EQUATIONS iii LIST OF FIGURES iv LIST OF TABLES vii Chapter 1: Literature study 1.1 1.1 Introduction 1.1 1.2 Wildebeest distribution and habitat 1.3 1.3 Past and present population status of the black wildebeest 1.4 1.4 Hybridization 1.6 1.5 Hybridization between blue and black wildebeest 1.8 1.6 A summary of the methods available for hybrid identification 1.11 1.6.1 External morphological characterization 1.11 1.6.2 Osteology 1.12 1.6.3 Molecular markers 1.12 1.6.3.1 Mitochondrial DNA sequencing 1.13 1.6.3.2 Y-chromosome markers 1.13 1.6.3.3 Single nucleotide polymorphisms 1.14 1.6.3.4 Microsatellite markers 1.15 1.6.4 Statistical analysis 1.16 1.6.4.1 Maximum likelihood methods 1.17 1.6.4.2 Bayesian methods 1.18 1.6.5 Case studies of hybrid detection 1.19 1.7 Aims 1.31 TABLE OF CONTENTS Chapter 2: Screening black wildebeest populations for putative hybrids 2.1 2.1 Introduction 2.1 2.2 Study Populations 2.2 2.2.1 Black wildebeest populations 2.2 2.2.2 Reference populations 2.6 2.2.3 Sampling 2.7 2.3 Methods for molecular analysis 2.8 2.3.1 DNA extraction and quantification 2.8 2.3.2 Amplification of microsatellite loci 2.10 2.3.3 Populations sampled 2.12 2.4 Statistical Analysis 2.13 2.4.1 Data organization 2.13 2.4.2 Genetic diversity 2.13 2.4.3 Hardy-Weinberg equilibrium (HWE) 2.14 2.4.4 Degree of genetic differentiation (FST) between populations 2.14 2.4.5 Analysis of Molecular Variance (AMOVA) 2.14 2.4.6 Assignment tests 2.15 2.5 Results and Discussion 2.17 2.5.1 Genotyping 2.17 2.5.2 Statistical Analysis 2.18 2.5.2.1 Genetic Diversity 2.18 2.5.2.2 Hardy-Weinberg Equilibrium (HWE) 2.21 2.5.2.3 Species-specific alleles 2.22 2.5.2.4 Degree of genetic differentiation (FST) and gene flow (Nm) between the Populations 2.25 2.5.2.5 Analysis of Molecular Variance (AMOVA) 2.28 2.5.2.6 Individual Assignment 2.30 TABLE OF CONTENTS Chapter 3: Application of molecular techniques to a known hybrid population 3.1 3.1 Application of molecular techniques 3.1 3.2 Molecular analysis 3.2 3.3 Results 3.2 3.4 Discussion 3.4 Chapter 4: Simulation study 4.1 4.1 Introduction 4.1 4.2 Case studies 4.1 4.3 Simulation study for wildebeest 4.4 4.3.1 Results and Discussion 4.6 Chapter 5: Grobler et al. 2005 revisited 5.1 5.1 Introduction 5.1 5.2 Reanalysis of microsatellite data 5.2 5.3 Results and Discussion 5.4 Chapter 6: Discussion 6.1 6.1 Genetic diversity in black wildebeest populations 6.1 6.2 Introgression of blue wildebeest alleles into black wildebeest populations 6.1 6.3 Recommendations for the future management of black wildebeest populations 6.3 Chapter 7: Summary 7.1 Chapter 8: Opsomming 8.1 Chapter 9: References 9.1 TABLE OF CONTENTS Appendix A: Sample list: Black wildebeest test populations Appendix B: Sample list: Reference populations Appendix C: Genetic profiles: Black wildebeest test populations Appendix D: Genetic profiles: Reference populations Appendix E: GeneClass Assignment test results LIST OF ABBREVIATIONS AND SYMBOLS LIST OF ABBREVIATIONS AND SYMBOLS Symbols: C Degrees Celsius % Percentage µ Micro: 10-6 ™ Trademark ® Registered Trademark Abbreviations: A Average number of alleles per locus A260/A280 Ratio of absorbency measured at 260 nm and 280 nm ABI Applied Biosystems AMOVA Analysis of Molecular Variance B.P. Before Present BP1 First generation backcrossed with parental species 1 BP2 First generation backcrossed with parental species 2 DETEA Department of Economic Development, Tourism and Environmental Affairs DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid dNTP Deoxynucleotide Triphosphate EDTA Ethylenediamine tetra-acetic acid F Forward primer F1 First generation F2 Second generation FST Genetic differentiation among populations g Gravitational Force Ho Observed heterozygosity Hz Unbiased heterozygosity HWE Hardy-Weinberg Equilibrium IUCN International Union for the Conservation of Nature and Natural Resources i LIST OF ABBREVIATIONS AND SYMBOLS K Number of clusters/populations km kilometres MCMC Markov Chain Monte Carlo mg Milligram Mg 2+ Magnesium ion MgCl2 Magnesium chloride min Minutes mM Millimolar mtDNA Mitochondrial Deoxyribonucleic Acid N Number of individuals N/A Not applicable ng Nanogram ng/μl Nanogram per micro litre nm nanometers Nm Gene flow No. Number NR Nature Reserve P1 Parental species 1 P2 Parental species 2 PCR Polymerase Chain Reaction R Reverse primer SD Standard deviation Sec seconds SNP Single Nucleotide Polymorphism SRY Sex-determining region Y-chromosome STR Short Tandem Repeat Ta Annealing temperature Tm Melting temperature U Units μl Micro litre µM Micromolar U.S. United States U.S.A. United States of America ii LIST OF EQUATIONS LIST OF EQUATIONS No. Title Page 2.1 Beer-Lambert equation 2.9 2.2 Modified Beer-Lambert equation 2.10 2.3 Equation for calculating gene flow 2.14 iii LIST OF FIGURES LIST OF FIGURES No. Title Page 1.1 Maps of sub-Saharan Africa showing (a) the historic distribution of blue 1.4 wildebeest and (b) black wildebeest. Historic and fossil records indicated by the broken line in (a) suggest a substantial overlap with black wildebeest (after Brink, 2005; adapted from Kingdon, 1997) 1.2 F1 hybrid animal (Photo: KwaZulu-Natal wildlife) 1.9 2.1 Sampling locations of black wildebeest herds in the Free State Province. 2.3 These localities are: Koppiesdam Nature Reserve (1) in the Northern Free State; Seekoeivlei Nature Reserve (2) stretching from Memel to Villiers; Willem Pretorius Nature Reserve (3) approximately 150 km north-east of Bloemfontein; Sterkfonteindam Nature Reserve (4) near Harrismith; Rustfonteindam Nature Reserve (5) east of Bloemfontein; Maria Moroka Nature Reserve (6) at the foot of the Thaba’Nchu mountains; Caledon Nature Reserve (7) located between Wepener and Smithfield; Reddersburg (8); Tussen-die-Riviere Nature Reserve (9) the southernmost reserve in the Free State Province; Gariepdam Nature Reserve (10) near Colesburg; Soetdoring Nature Reserve (11) and De Brug (12) just outside Bloemfontein and Erfenisdam Nature Reserve (13) located between Theunissen and Windburg (Image: http://maps.google.com) 2.2 Wildebeest translocations among Free State Nature Reserves until 2005 (TDR 2.5 – Tussen-die-Riviere, WPW – Willem Pretorius, NR – Nature Reserve) 2.3 Localities of new reference populations. Pure blue wildebeest were sampled 2.6 from Kruger National Park (1) and Kgalagadi Transfrontier National Park (3). Pure black wildebeest were sampled from Benfontein game farm (2) and Grootte Schuur game farm (4) (Image: http://maps.google.com) iv LIST OF FIGURES 2.4 Plot for detecting the number of groups (K) that best fit the data when both loci 2.30 were considered. The peak observed at K = 5 indicated that this is the true number of groups for this dataset (from STRUCTURE HARVESTER) 2.5 Plot of the mean likelihood L per K and variance per K value, obtained from 2.31 STRUCTURE on the dataset containing 21 populations genotyped for two polymorphic loci. The graph includes standard deviation to display likelihood variance (from STRUCTURE HARVESTER) 2.6 A graphical representation of the assignment results for five clusters, with data 2.33 from both loci 2.7 Plot for detecting the number of groups (K) that best fit the data. Only data for 2.33 the locus BM1824 was included (from STRUCTURE HARVESTER) 2.8 Plot of the mean likelihood L(K) and variance per K value obtained from 2.34 STRUCTURE on the dataset containing 21 populations genotyped for only the locus BM1824 (from STRUCTURE HARVESTER) 2.9 A graphical representation of the assignment of all the populations to two 2.36 different clusters, using data from only BM1824 3.1 Two F1 black wildebeest hybrids (Photo by Prof.