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Assessment of Species Boundaries of the Moringa Ovalifolia

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Assessment of Species Boundaries of the Moringa Ovalifolia ASSESSMENT OF SPECIES BOUNDARIES OF THE MORINGA OVALIFOLIA (DINTER & A. BERGER) IN NAMIBIA USING NUCLEAR ITS DNA SEQUENCE DATA A MINI-THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIODIVERSITY MANAGEMENT PROGRAMME OF THE UNIVERSITY OF NAMIBIA BY MARTHA KASIKU HAUSIKU 200125079 APRIL 2019 MAIN SUPERVISOR: Dr. E. Kwembeya (Department of Biological Sciences, University of Namibia) CO-SUPERVISOR: Professor. P. M. Chimwamurombe (Department of Natural and Applied Sciences, Namibia University of Science and Technology) ABSTRACT The genus Moringa comprises of thirteen (13) species of trees and shrubs, widely distributed in the tropical and subtropical regions. For its size, this genus is one of the phenotypically varied group of angiosperms whose members are mostly sought after for nutritional, nutraceutical and industrial uses. The aim of this study was to assess the species boundaries of Moringa ovalifolia in Namibia by using the nuclear Internal Transcribed Spacer (ITS) sequenced data analysis. A total of seventeen (17) edited sequences of M. ovalifolia together with thirteen (13) other sequences of Moringa retrieved from NCBI were used to construct phylogenetic trees in Molecular Evolutionary Genetic Analysis software version 6.0 (MEGA). The phylogenetic trees were resolved using Neighbor-joining method, Maximum Parsimony method, Maximum Likelihood method based on the Tamura-Nei and the bootstrap consensus tree was determined from 500 replicates that represented the evolutionary history of the taxa analyzed. The nuclear ITS molecular marker successfully delimited species boundaries of M. ovalifolia specimens that were investigated in this study. The topology of all three phylogenetic trees generated by the three methods were similar. All sequences of M. ovalifolia under investigation and those retrieved from NCBI) were grouped together and are not nested with other Moringa species. The different populations of the Namibian M. ovalifolia are not phylogenetically different though geographically isolated. This means that the Namibian M. ovalifolia is a taxonomically ‘good species’. The study demonstrated the useful role of ITS in determining the species boundaries of populations. It is recommended that efforts be made to initiate domestication and breeding programs of the Namibian M. ovalifolia. Key words: Specimens, Internal Transcribed Spacer, Moringa ovalifolia, species boundaries, phylogenetic trees. ii TABLE OF CONTENTS ABSTRACT ...................................................................................................................... ii ABBREVIATIONS ........................................................................................................... v LIST OF FIGURES .......................................................................................................... vi LIST OF TABLES .......................................................................................................... vii ACKNOWLEDGEMENTS ........................................................................................... viii DEDICATIONS ................................................................................................................ ix CHAPTER1: INTRODUCTION ....................................................................................... 1 1.1. Orientation of the study.................................................................................................. 1 1.2. Statement of the problem ............................................................................................... 4 1.3. Overall aim of the study ................................................................................................. 5 1.4. Specific objectives of the study ..................................................................................... 5 1.5. Null Hypotheses of the study ......................................................................................... 5 1.6. Significance of the study ................................................................................................ 6 1.7. Limitations of the study ................................................................................................. 7 CHAPTER 2: LITERATURE REVIEW ........................................................................... 8 2.1. Background ......................................................................................................................... 8 2.2. Uses of Moringa ............................................................................................................... 11 2.3. Geographic distribution of Moringa ovalifolia ................................................................. 13 2.4. Species concepts ............................................................................................................... 18 2.5. Delimitation of species boundary ..................................................................................... 19 2.6. Molecular phylogenetics ................................................................................................... 22 2.7. Phylogenetic tree reconstruction ....................................................................................... 25 2.8. Methods used to infer phylogenetic trees.......................................................................... 28 Rooting phylogenetic trees ...................................................................................................... 32 Statistical support for phylogenetic trees ................................................................................. 33 CHAPTER 3: MATERIALS AND METHODS ............................................................. 35 3.1. Research design ................................................................................................................ 35 3.2. POPULATION ................................................................................................................. 35 3.3. SAMPLE ........................................................................................................................... 35 iii 3.4 Genomic DNA extraction .................................................................................................. 35 3.5 ITS amplification protocol ................................................................................................. 37 3.6. Data collection .................................................................................................................. 38 3.7. Data analysis ..................................................................................................................... 38 3.7.1. DNA sequence generation ................................................................................... 38 3.8. Multiple Sequence Alignment: ................................................................................ 39 3.9. Methods of sequence analysis: distance, parsimony and ML .................................. 39 3.10. Rooting tree .......................................................................................................... 40 3.11. Research Ethics ........................................................................................................ 40 CHAPTER 4: RESULTS ................................................................................................. 41 4.1. Genomic DNA extraction ................................................................................................. 41 4.2. ITS PCR amplification ...................................................................................................... 42 4.3. Molecular phylogenetic analyses ...................................................................................... 43 4.3.1 Maximum Parsimony method ..................................................................................... 44 4.3.2. Maximum Likelihood method ................................................................................... 47 4.3.3. Neighbor-joining method ........................................................................................... 49 CHAPTER 5. DISCUSSION ........................................................................................... 52 CHAPTER 6: CONCLUSION ......................................................................................... 55 CHAPTER 7: RECOMMENDATIONS .......................................................................... 57 , CHAPTER 8: REFERENCES ....................................................................................... 58 CHAPTER 9: APPENDICES .......................................................................................... 80 APPENDIX A: Main ingredients for the CTAB protocol ....................................................... 80 APPENDIX B: Microwave instructions for agarose preparation of gel concentrations ≤2% w/v for electrophoresis ............................................................................................................. 82 iv ABBREVIATIONS BLAST- Basic Local Alignment Search Tool bp – Base pair CTAB- cetyl trimethylammonium bromide EDTA – Ethylenediaminetetra acetic acid DNA – Deoxyribonucleic acid ITS – Internal Transcribed Spacer regions PCR – Polymerase Chain Reaction RAPD – Random Amplified Polymorphic DNA RNA - Ribonucleic acid Taq- Thermophillus aquaticus oC – Degree Celsius µl – microliter v LIST OF FIGURES Figure 1: Map showing the global distribution of Moringa species l ..............................
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