MOLECULAR POLYMORPHISMS FOR PHYLOGENY, PEDIGREE AND POPULATION STRUCTURE STUDIES by YEAN WANG UNIVERSITY OF SYDNEY SCHOOL OF BIOLOGICAL SCIENCES NSW, AUSTRALIA A thesis submitted in the fulfillment of the requirements for the degree of Doctor of Philosophy December 2006 Declaration This thesis is presented in fulfillment of the requirements for the degree of Doctor of Philosophy. I hereby declare that the work demonstrated in this thesis is my own unless indicated otherwise. No part of this work has been submitted for any degree or diploma. The microsatellite isolation in Bactrocera tryoni reported in Chapter 6 of this thesis was done jointly with MSc student Hong Yu as part of her project. This thesis may be made available for reference from the university library and may be photocopied or loaned to other libraries. Yean Wang December 2006 TABLE OF CONTENTS LIST OF FIGURE................................................................................................. V LIST OF TABLE ................................................................................................. VII Acknowledgements............................................................................................. IX Abstract............................................................................................................... XI Abbreviations ..................................................................................................... XV Chapter 1..............................................................................................................1 Introduction ...........................................................................................................1 1.1 Review of DNA polymorphisms .............................................................................. 2 1.1.1 Mitochondrial DNA........................................................................................... 2 1.1.2 Microsatellites.................................................................................................... 6 1.1.3 The Y chromosome.......................................................................................... 12 1.1.4 Other molecular polymorphic markers............................................................ 14 1.1.5 Sex specific molecular polymorphisms ........................................................... 16 1.1.6 Incongruencies between polymorphism types................................................. 18 1.2 The present study.................................................................................................... 20 1.2.1 Aims of the research ........................................................................................ 20 1.2.2 mtDNA and phylogeny.................................................................................... 20 1.2.3 Paternity testing and pedigree construction..................................................... 22 1.2.4 Population analysis within species or between sibling species ....................... 22 1.3 An outline of the thesis ........................................................................................... 23 Chapter 2............................................................................................................25 Complete mtDNA sequence of the green monkey..............................................25 2.1 Introduction............................................................................................................. 25 2.2 Materials and Methods............................................................................................ 27 2.2.2 mtDNA sequencing.......................................................................................... 27 2.2.2.1 Normal and long PCR............................................................................... 27 2.2.2.2 Cloning the heteroplasmic region............................................................. 30 2.2.2.3 Sequencing............................................................................................... 32 2.3 Results..................................................................................................................... 33 2.3.1 Complete mtDNA sequence ............................................................................ 33 2.3.2 Heteroplasmic control region........................................................................... 37 2.4 Discussion............................................................................................................... 43 2.4.1 Complete mtDNA comparison among primates.............................................. 43 2.4.2 Heteroplasmy in the control region of primate mtDNA.................................. 50 Chapter 3............................................................................................................52 Analysis of phylogeny of Cercopithecinae using complete mitochondrial DNA sequence ............................................................................................................52 3.1 Introduction............................................................................................................. 52 3.1.1 Nomenclature of the green monkey…………………………………………..52 3.1.2 Phylogenetic analysis of the primate…………………………………………56 3.2 Materials and Methods............................................................................................ 58 I 3.2.1 Data source ...................................................................................................... 58 3.2.2 Phylogeny reconstruction among the species of primates............................... 59 3.3 Results and discussion ............................................................................................ 59 3.3.1 Phylogenetic analysis using complete mtDNA among primates..................... 59 3.3.2 Taxonomic level of Chlorocebus sabaeus and C. aethiops............................. 62 3.3.3 Phylogenetic analysis using single genes of mtDNA among primates ........... 62 3.3.4 Conclusion ....................................................................................................... 68 Chapter 4............................................................................................................71 mtDNA SNPs detection and St Kitts monkey verification by 12S rRNA gene .....71 4.1 Introduction............................................................................................................. 71 4.2 Materials and Methods............................................................................................ 72 4.2.1 PCR.................................................................................................................. 72 4.2.2 Single nucleotide polymorphisms (SNP) screening using dHPLC ................. 73 4.2.3 Sequence and haplotype analysis of SNPs ...................................................... 74 4.2.4 12S rRNA analysis of geographical origin of St Kitts monkeys ..................... 75 4.3 Results..................................................................................................................... 75 4.3.1 SNP identification by dHPLC.......................................................................... 75 4.3.2 Verification of species of the St Kitts green monkey by 12S rRNA gene....... 79 4.4 Discussion............................................................................................................... 81 Chapter 5............................................................................................................83 Paternity and pedigree analysis using cross-amplified microsatellites in green moneys ...............................................................................................................83 5.1 Introduction............................................................................................................. 83 5.1.1 Vervet Monkey Research Colony.................................................................... 83 5.1.2 Paternity assignment........................................................................................ 84 5.1.3 Microsatellite cross-amplification ................................................................... 85 5.2 Materials and methods............................................................................................ 87 5.2.1 DNA isolation.................................................................................................. 87 5.2.1.1 Autopure DNA extraction ........................................................................ 89 5.2.1.2 Manual DNA extraction........................................................................... 90 5.2.2 PCR amplification and genotyping.................................................................. 90 5.2.3 Data analysis.................................................................................................... 93 5.3 Results..................................................................................................................... 94 5.3.1 DNA extraction yield....................................................................................... 94 5.3.2 Cross-amplification.......................................................................................... 95 5.3.3 Maternal mismatch pedigree errors ................................................................. 96 5.3.4 Hardy-Weinberg Equilibrium........................................................................ 101 5.3.5 Paternity analysis ..........................................................................................