ASSESSING DIVERSITY OF Solanum nigrum L GROWN IN KENYA Rwigi Susan Wagio (B.Ed. Science) Reg. No. I56/CE/22421/2010 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University November, 2016 ii DECLARATION This thesis is my original work and has not been presented for a degree in any other university for any other award. Signature ……………………………………Date………………….. SUPERVISORS We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Dr Steven Runo Department of Biochemistry and Biotechnology Kenyatta University P.O. Box 43844-00100 Nairobi Signature …………………………………….Date……………………. Dr Alice Muchugi World Agroforestry Centre P. O. Box 30677-00100 Nairobi Signature………………………………………Date……………………… iii DEDICATION I dedicate this thesis to the Rwigi’s family. iv ACKNOWLEDGEMENTS I take this chance to thank my supervisors Dr. Steven Runo and Dr. Alice Muchugi for their guidance, contribution and supervision from the start of this research to the end. Dr Runo I will forever remain grateful for the tireless guidance and patience during my project work, may God bless your path. My sincere gratitude to all my Lecturers in the Department of Biochemistry and Biotechnology for taking me through the course work. Collective acknowledgments to my colleagues in The Plant Transformation Laboratory at Kenyatta University. I particularly acknowledge Mr. Erick Kuria, Mr. Nzaro, Joel Masanga, Mutero Ngure and Muli for their advice and support throughout the time I was working in the laboratory. Liz Njuguna you are awesome. The farmers who assisted me in Makuyu in Murang’a county, Kipkaren in Nandi county, Mauche in Nakuru county and Matanya in Laikipia county you are part of my success. I am greatly indebted to the Rwigi’s family. My brother David, my sisters Helen, Esther and Eve, my nephews Nickson and Simon who stood with me all through. I thank my beloved Kamau for his understanding and care during the long journey. Thanks for taking care of our kids while I was away. My children Calvin and Quinter you gave me the peace that I most needed. Lastly I wish to thank the Almighty Father for the gift of life, health and peace without which this work would not have been successful. To God be the glory. v TABLE OF CONTENTS DECLARATION ................................................................................................... ii DEDICATION...................................................................................................... iii ACKNOWLEDGEMENTS .................................................................................. iv TABLE OF CONTENTS ....................................................................................... v LIST OF TABLES .............................................................................................. viii LIST OF FIGURES .............................................................................................. ix ABBREVIATIONS AND ACRONYMS ............................................................... x ABSTRACT ......................................................................................................... xi CHAPTER ONE .................................................................................................... 1 INTRODUCTION ................................................................................................. 1 1.1 Background information................................................................................... 1 1.2 Problem statement and Justification ................................................................. 3 1.3 Hypotheses...................................................................................................... 4 1.4 Objectives’ ....................................................................................................... 5 1.4.1 General objective .......................................................................................... 5 1.4.2 Specific objectives’ ....................................................................................... 5 CHAPTER TWO ................................................................................................... 6 LITERATURE REVIEW ....................................................................................... 6 2.1 Solanum nigrum L ............................................................................................ 6 2.2 Taxonomy ........................................................................................................ 6 2.3 Biology ............................................................................................................ 7 2.4 Distribution and origin ................................................................................... 10 2.5 Economic importance ..................................................................................... 11 2.6 Constraints to production ............................................................................... 16 2.7 Genetic diversity of Solanum nigrum ............................................................. 16 2.8 Use of Markers to Determine Diversity ........................................................ 18 2.8.1 Restriction Fragment Length Polymorphism (RFLP) ................................... 20 vi 2.8.2 Amplified Fragment Length Polymorphism (AFLP) .................................... 21 2.8. 3 Random Amplified Polymorphic DNA (RAPD) ......................................... 21 2.8.4 Randomly Amplified Microsatellite Polymorphism (RAMP) ...................... 22 2.8.5 Simple Sequence Repeats (SSR) Markers .................................................... 23 2.8.6 SSR principles ............................................................................................. 25 2.8.7 Application of SSR markers ........................................................................ 25 CHAPTER THREE ............................................................................................. 28 MATERIALS AND METHODS ......................................................................... 28 3.1 Collection sites .............................................................................................. 28 3.1.1 Site selection ............................................................................................... 28 3.1.2 Sampling criteria ......................................................................................... 29 3.2 Morphological characterization ...................................................................... 31 3.3 DNA extraction .............................................................................................. 31 3.4 SSR marker analysis ...................................................................................... 32 3.5 Data analysis .................................................................................................. 34 3.6 Correlation of morphological and genetic data ............................................... 34 CHAPTER FOUR ............................................................................................... 35 RESULTS............................................................................................................ 35 4.1 Morphological characteristics ......................................................................... 35 4.2 Genetic characterization ................................................................................. 38 4.2.1 Genetic diversity estimates .......................................................................... 39 4.2.2 Partitioning genetic variation ...................................................................... 41 4.2.3 Population cluster analysis .......................................................................... 41 4.3 Correlation of morphological characters and genetic data .............................. 44 CHAPTER FIVE ................................................................................................. 47 DISCUSSION, CONCLUSION AND RECOMMENDATIONS.......................... 47 5.1 DISCUSSION ................................................................................................ 47 5.2 CONCLUSION .............................................................................................. 53 5.3 RECOMMENDATIONS ............................................................................... 54 vii REFERENCES .................................................................................................... 55 viii LIST OF TABLES Table 3. 1: Assessed morphological characteristics in S. nigrum populations............... 31 Table 3. 2: Primers used in the study .......................................................................... 33 Table 4. 1: Mean number of S. nigrum with observed characteristics. .......................... 37 Table 4. 2: SSR markers used, number of allele and annealing temperature ................. 38 Table 4. 3: Allele frequency, allele number, genetic diversity and PIC for SSR primers used in DNA amplification ........................................................... 39 Table 4. 4: Total band patterns, % polymorphism and Mean heterozygosity of S. nigrum collected from four study areas. .................................................... 40 Table 4. 5: Analysis of molecular variance (AMOVA) within and among S. nigrum population ................................................................................................