INTRASPECIFIC GENETIC VARIATION AMONG POPULATIONS OF KENYAN AFRICAN VIOLET. SAINTPAULIA RUPICOLA B. L. BURTT: A MOLECULAR APPROACH ^ r n:r BY SIMON MAIN A MACHARIA B.Ed Science (Egerton) A thesis submitted in partial fulfilment of the award of the degree of Master of Science in Botany mar DEPARTMENT OF BOTANY UNIVERSITY OF NAIROBI FEBRUARY, 1998 11 DECLARATION Declaration by the candidate I hereby declare that this thesis has not been submitted, either in the same or different form, to this or any other university for a degree. No part of this thesis may be reproduced without the prior permission of the author and/or University of Nairobi. , C i ^ f 4 1 ^ Simon Maina Macharia Date Declaration by the supervisors This thesis has been submitted with our approval as university supervisors. Ms\ II P r o f . r. K. Mulviurna D a t e 1st supervisor Chairman W a** Department of Botany V' University of Nairobi P. O. Boxl30197, Nairobi Id i t Dr. Rashid A. Aman Date 2nd supervisor Director, Research and Scientific Affairs National Museums of Kenya P. O. Box 40658, Nairobi DEDICATION To my mother, for your endless support and encouragement. To my daughter. Rachel and my son, Vickie, who have provided the driving force IV Acknowledgements A number of people deserve special acknowledgment in view of the invaluable support they gave me during my M.Sc course. In particular, I would like to thank Dr. Rashid Aman of the Department of Molecular Genetics. National Museums of Kenya (NMK) for providing most of the supervision for this work, and in whose lab its bulk has been carried out. I am also greatly indebted to Mr. Timothy Pearce of the Plant Conservation Programme (PCP), NMK for providing the initial funding for this project. Special thanks also go to the Board of Postgraduate Studies. University of Nairobi, for recommending me for a DAAD scholarship, and DAAD for providing the scholarship. I would also like to thank Prof. T. K. Mukiama of the Department of Botany, University of Nairobi for supporting my scholarship offer, and providing part of the supervision for this project especially through the final stages of preparation of this thesis. I am greatly indebted to Mr. Benny Bytebier of the PCP, NMK. who assisted me in the development of this project and personally guided me successfully through the field survey and provided part of the supervision for this project. Special thanks also go to Prof. Dr. Wolfgang Hachtel of the Botanisches Institut der Universitat Bonn, Germany, for offering me a chance to work in his lab for six months. I am also greatly indebted to Prof. Eric Knox of Rutgers University, USA, during his tenure at the NMK, for a lot of invaluable advice throughout the course of the project, and for providing me with tobacco chloroplast DNA probes used in the initial survey of this project. A big thank you to the members of the Department of Molecular Genetics, NMK for the bulk of technical training and support and providing a conducive and enjoyable environment in which I successfully completed this project. Last but by no means least. I would like to convey my special thanks to my parents, relatives, friends and all those who contributed in any way towards the successful completion of this study for their unwavering love, patience and encouragement during the course of this study. And to my friend Ngige. thank you for assisting me in printing! VI TABLE OF CONTENTS Title ............................................................................................................................... i Declaration................................................................................................................... ii Dedication ..................................................................................................................iii Acknowledgements ................................................. iv Table of contents ....................................................................................................... vi List of figures ........................................................................................................ Xi List of ta b le s.............................................................................................................. xii List of p la te s............................................................................................................. xiii Symbols/Abbreviations............................................................................................. xiv A bstract................................................................................................................... xviii CHAPTER 1 INTRODUCTION, LITERATURE REVIEW AND STATEMENT OF THE PROBLEM ........................................................................................................... 1 1.0 Introduction..................................................................................... 1 1.0. 1 The African V iolets......................................................................... 2 1.0. 2 The African violet as an endangered sp ecies............................... 2 1.0. 3 Saintpaulia rupicola......................................................................... 3 1.0. 4 Conservation efforts at the National Museums of K enya............ 4 1.0. 5 Statement of the problem...................................................................... 6 1.0. 6 Specific objectives................................................................................. 6 1.0. 7 Questions to be addressed ....................................................................6 1.1 Literature review ................................................................................ 7 1.1.1 Classification status of the African V io le ts................................. 7 1.1.2 Genomes available for DNA analysis in plant and animal taxa . 7 vii Table of contents continued 1.1.2.1 Mitochondrial DNA (mtDNA) ..................................................... 8 1.1.2.2 The chloroplast genome .................................................................... 10 1.1.2.3 Ribosomal D N A ..................................................................................11 1.1.2.3.1 The Internal Transcribed Spacer (ITS).......................................... 13 1.1.2.3.2 Structure of the ITS region ........................................................... 14 1.2 DNA techniques for determining genetic variation in plants . 14 1.2.1 Restriction site analysis.................................................................. 14 1.2.2 Randomly amplified Polymorphic DNA sequences (RAPDs) . 17 1.2.3 Polymerase Chain Reaction (PCR) and DNA sequencing .... 18 CHAPTER 2 .........................................................................................................20 MATERIALS AND METHODS ....................................................................... 20 2.0 Collection of plant material from the field ............................................... 20 2.0. 1 Collection of Saintpaulia sp nov. samples from Kacharoroni ............. 22 2.0. 2 Collection of S. rupicola samples from Cha Simba .............................. 22 2.0. 3 Collection of Saintpaulia rupicola samples from Mwarakaya ............. 23 2.0. 4 Collection of Saintpaulia sp. nov. samples from Mwache ................... 23 2.1 Extraction of DNA ...................................................................................... 26 2.1.1 Isolation of total (genomic) DNA: The CTAB procedure...................... 26 2.1.2 Extraction of replicative form (RF) plasmid DNA from E. c o li.................27 2.2 Determination of the amount of the D N A ............................................... 28 2.3 CsCl purification of genomic D N A ................................................. 29 Table of contents continued 2.4 Agarose gel electrophoresis........................................................................ 30 2.5 PCR amplification or ITS region ............................................................. 31 2.6 Purification of the PCR product............................................................... 32 2.7 Cloning of purified PCR product ............................................................. 33 2.7.1 Selection of recombinant v e c to r............................................................... 34 2.7.2 Colour selection........................................................................................... 34 2.7.3 Selection by PCR amplification ............................................................... 34 2.7.4 Small-scale preparation of replicative form (RF) plasmid DNA ............................................................................................................ 35 2.8 DNA sequencing........................................................................................ 35 2.8.1 Sequencing of recombinant plasmid D N A ............................................... 35 2.8.2 Direct sequencing of the PCR p ro d u ct................................................... 36 2.9 Preparation of sequencing gel and electrophoresis................................. 36 3.0.0 Alignment and analysis of DNA sequences ........................................... 38 CHAPTER 3 ......................................................................................................... 39 RESULTS .............................................................................................................. 39 3.0 Extraction and quantification of genomic DNA ......................... 39 3.1