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Molecular and Morphological Identification of Plants Consumed MOLECULAR AND MORPHOLOGICAL IDENTIFICATION OF PLANTS CONSUMED BY YELLOW BABOONS IN AMBOSELI, KENYA RISPAH NYAMBURA NG’ANG’A BSc. (Biology), University of Nairobi I56/82866/2015 A thesis submitted in partial fulfilment of the requirement for the award of a degree in Master of Science in Genetics at the School of Biological Sciences in the University of Nairobi, Kenya. 2019 1 DECLARATION This is my original work, which has never been presented for a degree in any other university or institution. Rispah Nyambura Ng’ang’a, Signature: Date: 13- November -2019 School of Biological Sciences, University of Nairobi. The thesis has been submitted with the approval of my supervisors: Dr. Nelson O. Amugune, Signature: Date: 13- November -2019 School of Biological Sciences, University of Nairobi, Dr. Mercy Y. Akinyi, Signature: Date: 13- November -2019 Department of Animal Science, Institute of Primate Research, Professor Elizabeth A. Archie, Signature: Date: 13- November -2019 Department of Biological Sciences, University of Notre Dame, USA. i DEDICATION To my parents Silas and Gladwell Ng’ang’a; siblings Rahab Mwihaki, Simon Gachoya, Meshack Mwangi, and James Gaitho; and all my dear friends. I am beyond grateful for your support and encouragement throughout this research period. May God bless you all! ii ACKNOWLEDGEMENTS First and foremost, I would like to express my sincerest gratitude to my supervisors: Dr. Nelson Amugune, Dr. Mercy Akinyi, and Professor Elizabeth Archie, for their guidance during the entire period of this study. I am eternally grateful for always being available for consultations, and could not have imagined having better advisors for my dissertation. I am exceptionally grateful to the University of Nairobi for allowing me to pursue this Master’s degree through a scholarship. This opportunity has provided a great deal of exposure. I want to acknowledge the Amboseli Baboon Research Project team for allowing me to conduct my research with them. I am eternally grateful for this opportunity and financial support. I would also like to thank Professor Archie, Mr. Kinyua Warutere and Ms. Mauna Dasari, all of whom aided in the collection and preservation of the plant materials from the study site in Amboseli. I would also like to thank the Institute of Primate Research (IPR), Kenya, for allowing me to conduct my research at their laboratories. Additionally, I am very grateful to the barcoding team at the Molecular Genetics Laboratory, situated at the National Museums of Kenya, for their assistance and advice. Finally, I am exceedingly grateful to the Almighty God for giving me strength, guidance, and good health throughout this research period. iii TABLE OF CONTENTS DECLARATION ..................................................................................................... i DEDICATION ........................................................................................................ ii ACKNOWLEDGEMENTS .................................................................................. iii LIST OF TABLES ................................................................................................ vii LIST OF FIGURES ............................................................................................... ix LIST OF APPENDICES ....................................................................................... xi LIST OF ABBREVIATIONS AND ACRONYMS ............................................ xii CHAPTER ONE: INTRODUCTION ................................................................... 1 1.1 Background to the study ..................................................................................... 1 1.2 Problem statement .............................................................................................. 3 1.3 Justification of the study ..................................................................................... 3 1.4 Objectives ........................................................................................................... 4 1.4.1 General objective ......................................................................................... 4 1.4.2 Specific objectives ....................................................................................... 5 1.5 Hypotheses .......................................................................................................... 5 CHAPTER TWO: LITERATURE REVIEW ..................................................... 6 2.1 Plant diet analysis of yellow baboons ................................................................. 6 2.2 Traditional methods of analysing plant composition in diets ............................. 7 2.3 Genetic identification of plants ........................................................................... 8 2.4 Applications of DNA (meta)barcoding ............................................................ 11 2.4.1 Dietary analysis .......................................................................................... 11 2.4.2 Characterizing biodiversity ........................................................................ 11 2.5 Plant barcodes ................................................................................................... 12 2.5.1 Plastid-based markers ................................................................................ 12 2.5.1.1 Protein-coding barcodes ...................................................................... 12 2.5.1.2 Non-coding barcodes ........................................................................... 13 2.5.2 Nuclear-based markers ............................................................................... 16 2.6 DNA barcoding database .................................................................................. 18 iv TABLE OF CONTENTS CHAPTER THREE: MATERIALS AND METHODS .................................... 20 3.1 Study area ......................................................................................................... 20 3.2 Collection of plant materials ............................................................................. 22 3.2.1 Preparation of voucher specimens ............................................................. 24 3.3 Molecular analysis ............................................................................................ 25 3.3.1 DNA extraction .......................................................................................... 25 3.3.2 Agarose gel electrophoresis ....................................................................... 28 3.3.3 Polymerase chain reaction (PCR) .............................................................. 29 3.3.4 PCR product purification and sequencing ................................................. 31 3.4 Data analysis ..................................................................................................... 32 3.5 Species identification ........................................................................................ 33 3.5.1 Sequence similarity analysis ...................................................................... 33 3.5.2 Phylogenetic analysis ................................................................................. 34 CHAPTER FOUR: RESULTS ............................................................................ 37 4.1 Morphological identification ............................................................................ 37 4.2 Molecular analysis ............................................................................................ 59 4.2.1 DNA extraction .......................................................................................... 59 4.2.2 Polymerase chain reaction (PCR) .............................................................. 60 4.2.2.1 The amplification success rates of the candidate barcoding genes ..... 60 4.2.2.2 The amplification success rates in monocotyledons and dicotyledons ......................................................................................................................... 62 4.3 Sanger sequencing ............................................................................................ 69 4.3.1 Length distribution of sequences ............................................................... 69 4.4 Species identification ........................................................................................ 71 4.4.1 Sequence similarity analysis based on BLAST algorithm ......................... 71 4.4.1.1 The identification of monocotyledons based on BLAST analysis ...... 71 4.4.1.2 The identification of dicotyledons based on BLAST analysis ............ 74 4.4.2 Tree-based identification............................................................................ 77 v TABLE OF CONTENTS 4.4.2.1 The identification of monocotyledons based on phylogenetic analysis ......................................................................................................................... 87 4.4.2.2 The identification of dicotyledons based on phylogenetic analysis .... 88 4.5 Consensus identities based on morphological and molecular analyses ............ 90 CHAPTER FIVE: DISCUSSION ....................................................................... 94 5.1 Morphological identification ............................................................................ 94 5.2 Molecular analysis ............................................................................................ 94 5.3 Sanger sequencing ...........................................................................................
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