MOLECULAR GENETIC STUDIES ON COMMON CARP (CYPRINUS CARPIO L.) by Binh Thanh Thai (B. Sc., Grad. Dip) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Science and Primary Industries Faculty of Education, Health and Science Charles Darwin University, Casuarina, Northern Territory, Australia August, 2006 ii CHARLES DARWIN UNIVERSITY CANDIDATE DECLARATION I certify that the thesis entitled: Molecular genetic studies on common carp (Cyprinus carpio L.) Submitted as a thesis for the degree of Doctor of philosophy of the Charles Darwin University, is the result of my own investigations, and all references to ideas and work of other researches have been specifically acknowledged. I hereby certify that the work embodied in this thesis has not already been accepted in substance for any degree, and is not being currently submitted in candidature for any other degree. Full Name: Binh Thanh Thai Signed...................................................................................................... Date......................................................................................................... iii ACKNOWLEDGEMENTS I am very grateful to Australian Agency for International Development (AusAID) for financially supporting this research, and Charles Darwin University (CDU), Deakin University, Arafura Timor Research Facility (ATRF), Australia, and Research Institute for Aquaculture No 1 (RIA 1), Vietnam for providing facilities for my studies. I would like to thank my principal supervisor, Professor Christopher Austin, for his support, guidance, enthusiasm and encouragement during the entire course of PhD study. I feel extremely lucky to have Dr. Chris Austin as my major professor. I am grateful to Dr. Christopher Burridge (Associate Supervisor, Deakin University) for providing significant support for my research. I also need to express my thanks to Dr. Sarah Smith and Mark Schultz for their valuable suggestions and for proof reading my thesis. My research involved collecting common carp samples from many different countries around the world. I would like to take this opportunity to thank: Prof. Li Sifa (China), Dr. Varadi Laszlo, Dr. Zsigmond Jeney (Hungary), Dr. Y. Basavaraju (India), Dr. Graham C. Mair (AIT-Thailand), Dr. Ilan Karplus, Dr. Uri Lavi (Israel), Dr. Estu Nugroho (Indonesia), Dr. Dunz D.N. (Vietnam), Ms. Yuko Lemura (Japan), Lukas Kalous (Czech public), Dr. Martin Kumar, Dr. Matthew Baranski, Vince Versace (Australia), Dr. Riho Gross (Estonia), Phuc Dinh Phan iv (Research Institute for Aquaculture No III), Van Si Ngo, Hung Quang Le, Ninh Huu Nguyen, Tan Thi Nguyen, Anh The Bui (RIA1), staff of RIA1, Yen Thuy Duong (Can Tho University, Vietnam), and numerous fish farmers in Vietnam, all of whom helped me to collect the samples. I would like to thank Dr. Tuan Anh Pham for his support and encouragement for my study. Special thanks are given to Dr. Martin Kumar of South Australian Research and Development Institute (SARDI) for his support and advice throughout this study. I give my sincere thanks to Dr. Kohji Mabuchi for his providing mitochondrial DNA data of Lake Biwa common carp from Japan. I wish to thank my colleagues, both past and present, from the Molecular Ecology and Biodiversity Laboratory, who provided me with various assistance. In particular, Mark Schultz, Mike Truong, Leon Meggs, Sally Williamson, Claire McClusky, Amanda Peucker, Rebecca Overeem, Dr. Matthew Baranski, Celeste Jacq, Dr. Nicholas Murphy and Dr. Adam Miller have assisted me greatly with laboratory techniques. I wish to thank Long Nguyen, Bac Nguyen, Hanh Nguyen, Nhu Ly and Luong Van Thing for their encouragement and support for my study while I studied in Australia. v Finally, I would like to thank my family in Vietnam for allowing me to be far away from home. Love and care from my parents (Ung Doan Thai, Ke Thi Nguyen), brothers (An Thanh Thai, Toan Thai), sisters (Phuc Hong Thi Thai, Ha Thanh Thai), sister in-law (Thoa Kim Nguyen), neice Linh Phuong Thi Thai, and nephew Nam Phuong Thai have been critical for the success of my studies. vi CONTENTS ACKNOWLEDGEMENTS ................................................................................................. iii CONTENTS .......................................................................................................................... vi LIST OF FIGURES .............................................................................................................. ix LIST OF TABLES ................................................................................................................ xi ABSTRACT ......................................................................................................................... xiii CHAPTER 1 GENERAL INTRODUCTION .............................................................................................. 1 1.1 COMMON CARP ......................................................................................................................... 1 1.1.1 Distribution, taxonomy and translocation ............................................................... 1 1.1.2 Biology of common carp ......................................................................................... 3 1.1.3 Aquaculture ............................................................................................................. 4 1.1.4 Common carp culture in Vietnam ........................................................................... 5 1.2 MOLECULAR MARKERS ......................................................................................................... 8 1.2.1 Molecular techniques .............................................................................................. 8 1.2.1.1 Mitochondrial DNA Sequencing ..................................................................... 9 1.2.1.2 Single Strand Conformation Polymorphism (SSCP) ..................................... 10 1.2.1.3 Microsatellites ................................................................................................ 11 1.2.2 Applications of molecular markers ....................................................................... 12 1.2.2.1 Population genetics ........................................................................................ 12 1.2.2.2 Aquaculture .................................................................................................... 17 1.2.2.3 Conservation genetics .................................................................................... 21 1.2.2.4 Taxonomy and evolution ............................................................................... 22 1.2.2.5 Molecular genetic studies on common carp ................................................... 29 1.3 RESEARCH OBJECTIVES AND THESIS FORMAT .............................................................. 32 CHAPTER 2 USING MITOCHONDRIAL NUCLEOTIDE SEQUENCES TO INVESTIGATE DIVERSITY AND GENEALOGICAL RELATIONSHIPS WITHIN COMMON CARP (CYPRINUS CARPIO L.)* ................................................................................................... 35 2.1 INTRODUCTION ...................................................................................................................... 35 2.2 MATERIALS AND METHODS ................................................................................................ 37 2.2.1 Common carp samples .......................................................................................... 37 2.2.2 DNA extraction and Amplification of Mitochondrial DNA ................................. 37 2.2.3 Data Analysis and Phylogenetic Reconstruction .................................................. 38 2.3 RESULTS ................................................................................................................................... 39 2.4 DISCUSSION ............................................................................................................................. 41 CHAPTER 3 MOLECULAR TAXONOMY AND EVOLUTION OF COMMON CARP ................... 47 3.1 INTRODUCTION ...................................................................................................................... 47 3.2 MATERIALS AND METHODS ................................................................................................ 51 3.2.1 Sample collection .................................................................................................. 51 3.2.2 DNA Extraction and Amplification ...................................................................... 51 3.2.3 Phylogenetic Analyses .......................................................................................... 52 vii 3.2.4 Phylogenetic hypothesis testing ............................................................................ 53 3.3 RESULTS ................................................................................................................................... 54 3.3.1 Phylogenetic analysis ............................................................................................ 54 3.3.2 Test of taxonomic and evolutionary hypotheses ................................................... 56 3.4 DISCUSSION ............................................................................................................................. 57 3.4.1 Sequence variation ................................................................................................ 57 3.4.2 Phylogeny and evolutionary history .....................................................................