Population Genetics of Western Mediterranean Islands - Malta: a case study A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Josef Caruana List of Contents LIST OF CONTENTS .................................................................................................................... 2 LIST OF FIGURES: ....................................................................................................................... 7 LIST OF TABLES .......................................................................................................................... 9 LIST OF ABBREVIATIONS ..................................................................................................... 10 ABSTRACT ................................................................................................................................. 11 DECLARATION .......................................................................................................................... 12 COPYRIGHT STATEMENT ..................................................................................................... 13 DEDICATION ............................................................................................................................. 15 ACKNOWLEDGMENTS ............................................................................................................ 16 CHAPTER 1: INTRODUCTION .............................................................................................. 17 1.1: Maltese archaeology ............................................................................................................................17 1.2: The archaeological sites which were sampled ......................................................................................24 1.2.1: The Kercem site ..................................................................................................................................24 1.2.2: The Tal-Barrani and the Tal-Gardina sites ...........................................................................................26 1.3: Ancient DNA .........................................................................................................................................29 1.3.1: The study of ancient DNA and its authentication ................................................................................29 1.3.2: DNA degradation ................................................................................................................................33 1.4: Burial and storage conditions ...............................................................................................................33 1.5: Thermal age ..........................................................................................................................................35 2 1.6: Ancient DNA contamination .................................................................................................................36 1.7: Genetic markers and their use in population genetics .........................................................................37 1.8: Mitochondrial DNA (mtDNA) ................................................................................................................37 1.9: Y chromosome ......................................................................................................................................39 1.10: Analysing mtDNA and MSY sequences ...............................................................................................40 1.11: Mitochondrial DNA influences in Europe from the Near East .............................................................44 1.12: Possible refugia and areas of isolation in Europe ...............................................................................50 1.13: African mtDNA in the Mediterranean region......................................................................................54 1.14: The influence of North Africa on European populations ....................................................................55 1.15: Y-STR Studies in the Mediterranean ...................................................................................................59 1.16: The use of alternative markers in population genetic studies of regions neighbouring Malta ...........65 1.17: Aims of the study ................................................................................................................................72 CHAPTER 2: MATERIALS AND METHODS ........................................................................ 75 2.1: Acquiring permission to sample the inhumations from the Maltese burial sites .................................75 2.2: Obtaining the modern Maltese samples ...............................................................................................77 2.3: Obtaining the archaeologists’ samples .................................................................................................77 2.4: Sample conditions ................................................................................................................................78 2.5: Precautions and quality control ............................................................................................................79 2.6: Precautions in the laboratories.............................................................................................................81 2.7: Ethidium bromide precautions .............................................................................................................84 2.8: Sampling ...............................................................................................................................................84 2.9: DNA extraction methods ......................................................................................................................86 2.9.1: Bone preparation ...............................................................................................................................86 2.9.2: Removal of tooth dentine ...................................................................................................................87 3 2.9.3: DNA extraction and purification from bone and teeth ........................................................................88 2.9.4: Modern DNA extraction using chelex ..................................................................................................89 2.10: Polymerase chain reactions ................................................................................................................90 2.10.1: Ancient DNA markers used in the study ............................................................................................90 2.10.2: Amplification ...................................................................................................................................93 2.10.3: Modern mtDNA markers ..................................................................................................................95 2.11: Preparation of DNA size markers ........................................................................................................99 2.12: Gel electrophoresis .............................................................................................................................99 2.13: Comparison between routine reagents and new substitutes used in the laboratory ....................... 100 2.13.1: Comparison of electrophoresis buffers ........................................................................................... 100 2.13.2: Comparison between Gel Red and ethidium bromide ..................................................................... 101 2.14: Extraction of DNA from agarose gels ................................................................................................ 102 2.15: Direct PCR purification ...................................................................................................................... 103 2.16: Comparison between different kinds of silica gel spin columns ....................................................... 104 2.17: Cloning of the PCR products ............................................................................................................. 105 2.17.1: TOPO® cloning kit ............................................................................................................................ 105 2.17.2: StrataCloneTM PCR cloning kit ......................................................................................................... 109 2.18: Sex identification .............................................................................................................................. 110 2.19: Measurement of DNA concentration using Nanodrop ..................................................................... 112 2.20: Modern male genotyping ................................................................................................................. 113 2.21: Data Analysis .................................................................................................................................... 118 CHAPTER 3: ANCIENT DNA ANALYSIS ............................................................................ 123 3.1: Objectives of the aDNA study and the problems encountered during its excecution ........................ 123 3.2: Ancient DNA amplification and interpretation ..................................................................................