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GENETIC DIVERSITY and VIRULENCE STUDY of SEVEN Striga Hermonthica ECOTYPES from KENYA and UGANDA on SELECTED SORGHUM VARIETIES A

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GENETIC DIVERSITY and VIRULENCE STUDY of SEVEN Striga Hermonthica ECOTYPES from KENYA and UGANDA on SELECTED SORGHUM VARIETIES A GENETIC DIVERSITY AND VIRULENCE STUDY OF SEVEN Striga hermonthica ECOTYPES FROM KENYA AND UGANDA ON SELECTED SORGHUM VARIETIES KATAKA ATANDA JOEL (B. Ed Sc.) I56/CE/14403/2009 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. AUGUST, 2014 ii DECLARATION Declaration by the candidate This thesis is my original work and has not been presented for a degree in any other university or for any other award. Kataka Atanda Joel Signature……………………………… Date…………………… Declaration by the 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 Nairobi Signature…………………………. Date…………………………… Dr. Alice Muchugi International Centre for Research in Agroforestry P.O Box 30677, 00100 Nairobi Signature………………………… Date……………………………. iii DEDICATION This thesis is dedicated to my son Marques Atanda Kataka. iv ACKNOWLEDGEMENT First, I thank God the almighty for the good health he granted me during this period. I appreciate the support and encouragement from my supervisors, family members, friends and colleagues. May God bless you all. My deepest appreciation to Dr. Steven Runo for granting me the research project, his hands on supervision and his timely and adequate provision of the financial support that made the completion of this work a reality. I thank Dr. Alice Muchugi for her supervisory role, her keen interest in my work and for her unconditional support and guidance during this period. Specifically, I thank my uncle Francis Atwoli: Central Organization of Trade Unions (COTU) for paying my fees and supporting me financially during my masters study period. I sincerely appreciate the help, encouragement, moral support and advice provided to me by Dr. Allan Jalemba and Dr. Omwoyo Ombori. v TABLE OF CONTENTS DECLARATION ------------------------------------------------------------------------------ ii DEDICATION -------------------------------------------------------------------------------- iii ACKNOWLEDGEMENT ------------------------------------------------------------------ iv TABLE OF CONTENTS -------------------------------------------------------------------- v LIST OF TABLES --------------------------------------------------------------------------- vii LIST OF FIGURES ------------------------------------------------------------------------ viii ABBREVIATIONS AND ACRONYMS ------------------------------------------------- ix ABSTRACT ------------------------------------------------------------------------------------- x CHAPTER ONE INTRODUCTION 1.1 Background of the study ------------------------------------------------------------------ 1 1.2 Problem statement -------------------------------------------------------------------------- 4 1.3 Justification ---------------------------------------------------------------------------------- 5 1.4 Hypothesis ----------------------------------------------------------------------------------- 6 1.5 Objectives ------------------------------------------------------------------------------------ 6 1.5.1 General objectives ------------------------------------------------------------------------ 6 1.5.2 Specific objectives ----------------------------------------------------------------------- 6 1.6 Significance of the study ------------------------------------------------------------------ 7 CHAPTER TWO LITERATURE REVIEW 2.1 Origin, distribution and infestation of Striga to crops --------------------------------- 8 2.2 Striga biology ------------------------------------------------------------------------------- 9 2. 3 Economic importance of Striga weed ------------------------------------------------- 12 2.4 Importance of sorghum and the extent of infestation by Striga in SSA ---------- 15 2.5 Striga control and management --------------------------------------------------------- 16 2.6 Genetic diversity within and between S. hermonthica populations --------------- 18 2.7 Research achievements on S. hermonthica diversity -------------------------------- 20 2.8 Molecular markers used in diversity studies ------------------------------------------ 22 2.8.1 Morphological markers ------------------------------------------------------------- 23 2.8.2 Biochemical markers --------------------------------------------------------------- 23 2.8.3 DNA markers ------------------------------------------------------------------------ 24 2.8.3.1 Random Amplified Polymorphic DNA ------------------------------------- 25 2.8.3.2 Restriction Fragment Length Polymorphism ------------------------------ 26 2.8.3.3 Amplified Fragment Length Polymorphism ------------------------------- 27 2.8.3.4 Simple Sequence Repeats ----------------------------------------------------- 27 2.8.3.4.1 Development of SSR ----------------------------------------------------- 29 CHAPTER THREE MATERIALS AND METHODS 3.1 Genetic diversity of S. hermonthica ecotypes ---------------------------------------- 32 3.1.1 Plant materials ----------------------------------------------------------------------- 32 3.1.2 Seed pre-conditioning and culturing ---------------------------------------------- 33 vi 3.1.3 DNA extraction procedure --------------------------------------------------------- 34 3.1.4 DNA estimation and quantification ----------------------------------------------- 34 3.1.5 Polymerase chain reaction --------------------------------------------------------- 35 3.1.6 Fragment analysis ------------------------------------------------------------------- 36 3.1.7 Data analyses ------------------------------------------------------------------------- 36 3.2 Assesment of sorghum for their response to S. hermonthica infection ----------- 38 3.2.1 Plant materials ----------------------------------------------------------------------- 38 3.2.2 Sorghum seed germination --------------------------------------------------------- 38 3.2.3 Striga seeds conditioning ----------------------------------------------------------- 39 3.2.4 Infection of sorghum seedlings with S. hermonthica -------------------------- 39 3.2.5 Determination of S. hermonthica radicle attachment on roots of sorghum - 40 3.2.6 Phenotype of resistance------------------------------------------------------------- 40 3.2.7 Statistical analysis ------------------------------------------------------------------- 40 CHAPTER FOUR RESULTS 4.1 Genetic diversity of S. hermonthica ecotypes ---------------------------------------- 42 4.1.1 S. hermonthica seed germination frequency - Error! Bookmark not defined. 4.1.2 Fragment analysis ------------------------------------------------------------------- 43 4.2 Assessment of sorghum for their response to S. hermonthica infection ---------- 48 4.2.1 Transfer of sorghum seedlings to rhizotrons ------------------------------------ 48 4.2.2 S. hermonthica attachment and phenotype of resistance ---------------------- 49 4.2.3 Evaluation of susceptibility and resistance -------------------------------------- 50 4.2.4 Evaluation of S. hermonthica dry biomass, length and number ------------- 51 CHAPTER FIVE DISCUSSION, CONCLUSION AND RECOMMENDATIONS 5.1 Discussion --------------------------------------------------------------------------------- 54 5.1.1 Genetic diversity of S. hermonthica ecotypes -------------------------------------- 54 5.1.2 Assesment of sorghum for their response to S. hermonthica infection --------- 56 5.2 Conclusion --------------------------------------------------------------------------------- 60 5.3 Recommendations ------------------------------------------------------------------------ 60 REFERENCES ------------------------------------------------------------------------------- 61 APPENDICES -------------------------------------------------------------------------------- 70 Appendix 1: DNA extraction protocol ---------------------------------------------------- 70 vii LIST OF TABLES Table 2.1: Crops infested with Striga weeds in SSA and the degree of infestation .... 9 Table 2.2: Cereal production and consumption in kenya in 2009 ............................ 14 Table 2.3: Primer sets tested for use in diversity studies of S. hermonthica ........... 31 Table 3.1: Locations where S. hermonthica seeds were collected ............................ 32 Table 3.2: Sequences of oligonucleotide used to PCR amplify SSR. ....................... 35 Table 3.3: The country of origin of the sorghum genotypes used in the study ......... 38 Table 4.1: Germination frequency of the S. hermonthica seeds ............................... 43 Table 4.2: Summary data for each primer................................................................. 44 Table 4.3: Pairwise population matrix of Nei‟s genetic distance ............................. 45 Table 4.4: Analysis of molecular variance for S. hermonthica populations ............. 48 Table 4.5: The means of S. hermonthica plant numbers, dry biomass and length ... 53 viii LIST OF FIGURES Figure 2.1: Pictures of S. hermonthica, S. asiatica and S. gesnerioides showing variations in morphology ................................................................................... 10 Figure 2.2: Life cycle of Striga showing the major stages of development ............. 12 Figure 4.1: In vitro germination of S. hermonthica seeds 18 hours after introduction of germination stimulant (GR24) .................................................
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