UNIVERSITY OF NAIROBI. GENETIC DIVERSITY AND PHYLOGENETIC ANALYSIS OF Adansonia Spp. IN THE KENYA/TANZANIA TRANSECT. BY CATHERINE WAMBUI KAMAU I56/71711/2014 Center for Biotechnology and Bioinformatics College of Biological and Physical sciences The University of Nairobi A thesis submitted in partial fulfillment for the degree of Master of Science in Biotechnology in the Center for Biotechnology and Bioinformatics (CEBIB) University of Nairobi. November 2016 DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Catherine Wambui Kamau I56/71711/2014 University of Nairobi Signature: ____________________ Date __________________ APPROVED BY This thesis has been submitted for examination with our approval as supervisors Dr. Edward Muge Department of Biochemistry, University of Nairobi Signature____________________ Date_________________ Dr. George Obiero Director, Centre for Biotechnology and Bioinformatics University of Nairobi Signature____________________ Date_________________ Dr. Alice Muchugi, PhD Genebank Manager, Genetic Resources Unit, World Agroforestry Centre Signature____________________ Date________________ i DEDICATION To my family and dear friend, Louis Njora for their support and encouragement. ii ACKNOWLEDGEMENTS First and foremost, I want to thank the Almighty God, in whom I have found the strength, will, health and guidance to make this research a success. To my family and dear friends, I thank you for your love, prayers, encouragement and support. Secondly, I want to acknowledge my supervisors, Dr. Edward Muge and Dr. George Obiero, who have provided me with technical insights, critic and much needed guidance. This work would not have been possible without the support of my supervisor Dr. Alice Muchugi and the World Agroforestry Centre. Thank you so much for offering me this opportunity. I greatly appreciate Robert Kariba-ICRAF for guiding me every step of the way in my lab work and largely influencing the success of this work. I also appreciate Samuel Manthi and Vincent Njunge from ICRISAT for their technical insights and assistance which was pivotal in this work. I am greatly indebted to you all and may the good Lord bless you and give you prosperity in your future endeavors. I also wish to acknowledge my fellow colleagues from the Centre for Biotechnology and Bioinformatics for their much needed suggestions and comments. iii TABLE OF CONTENTS DECLARATION ............................................................................................................................ i ACKNOWLEDGEMENTS .......................................................................................................... iii TABLE OF CONTENTS .............................................................................................................. iv LIST OF TABLES ....................................................................................................................... vii LIST OF FIGURES .................................................................................................................... viii LIST OF APPENDICES ............................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................................ x ABSTRACT .................................................................................................................................. xi CHAPTER ONE ............................................................................................................................ 1 INTRODUCTION ......................................................................................................................... 1 1.1 Background .......................................................................................................................... 1 1.2 Problem Statement ............................................................................................................... 2 1.3 Justification of the Study ..................................................................................................... 3 1.4 Research questions ............................................................................................................... 4 1.5 Hypotheses ........................................................................................................................... 4 1.6 Study objectives ................................................................................................................... 4 1.6.1 Main objective .............................................................................................................. 4 1.6.2 Specific objectives ........................................................................................................ 4 CHAPTER TWO ........................................................................................................................... 5 LITERATURE REVIEW .............................................................................................................. 5 2.1 Origin and distribution of baobab ........................................................................................ 5 2.2 Floral morphology and pollination of baobab ...................................................................... 6 2.3 Common uses of Adansonia species .................................................................................... 6 2.4 The African species of Adansonia ....................................................................................... 7 2.4.1 The diploid progenitor of A. digitata ................................................................................ 8 2.5 Molecular Markers ............................................................................................................... 9 2.5.1 Microsatellite Markers .................................................................................................... 10 2.6 Phylogenetic inference of the African Adansonia species ................................................. 13 iv CHAPTER THREE ..................................................................................................................... 17 MATERIALS AND METHODS ................................................................................................. 17 3.1 Study areas and sampling ................................................................................................... 17 3.2 Seed germination ............................................................................................................... 17 3.3 DNA isolation .................................................................................................................... 17 3.4 DNA quantification and quality analysis ........................................................................... 18 3.5 Primer selection and optimization...................................................................................... 19 3.6 ITS Primers used to amplify the ITS region ...................................................................... 22 3.7 PCR amplification of SSRs ................................................................................................ 22 3.8 PCR amplification of ITS region ....................................................................................... 23 3.9 Quantification of PCR products ......................................................................................... 24 3.10 Purification of PCR products ........................................................................................... 24 3.11 Preparation of SSR amplification products for Capillary Electrophoresis ...................... 24 3.12 Microsatellite allele scoring and analysis ........................................................................ 25 3.13 Analysis of Molecular Variance (AMOVA) .................................................................... 25 3.14 Principal Coordinate Analysis (PCoA) ............................................................................ 26 3.15 Cluster analysis ................................................................................................................ 26 3.16 Measures of genetic diversity .......................................................................................... 26 3.17 Polymorphic Information Content (PIC) value determination ......................................... 26 3.18 Phylogenetic analysis of the African Adansonia species ................................................. 27 CHAPTER FOUR ........................................................................................................................ 28 RESULTS .................................................................................................................................... 28 4.0 DNA Extraction ................................................................................................................. 28 4.1 SSR Amplification ............................................................................................................. 28 4.2 Allele Scoring .................................................................................................................... 29 4.3 Analysis of Molecular Variance ........................................................................................ 30 4.4 Principal Coordinate Analysis ..........................................................................................