GENETIC BASIS OF Striga hermonthica (Del.) BENTH RESISTANCE IN WILD SORGHUM ACCESSIONS Dorothy Annah Mbuvi (BSc) I56/28368/2014 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Masters of Science (Biotechnology) in the School of Pure and Applied Science of Kenyatta University June, 2017 ii DECLARATION This thesis is my original work and has not been presented for a degree or other awards in any other university. Dorothy Annah Mbuvi Signature…………………… Date………………… Supervisors: We confirm that the candidate under our supervision carried out the work reported in this thesis. Dr. Steven Runo Department of Biochemistry and Biotechnology Kenyatta University P.O Box 43844-00100 Nairobi, Kenya Signature……………………. Date ……………………… Dr. Mark Wamalwa Department of Biochemistry and Biotechnology Kenyatta University P.O Box 43844-00100 Nairobi, Kenya Signature ……………………. Date……………………… iii DEDICATION I dedicate this thesis to my dear parents Paul and Margaret for their perseverance, prayers, comfort and encouragement accorded to me during my studies. iv ACKNOWLEDGMENTS I greatly appreciate my supervisors Dr. Steven Runo and Dr. Mark Wamalwa for the guidance they provided as I undertook my project. Your valuable time, ideas, productive discussions, instructive guidance and unlimited support during the research work are highly appreciated. To my dear parents, Paul and Margaret, my brother George and my sisters Mary and Ruth, I appreciate you for realizing the academic potential in me and encouraging me to further my studies. I am proud of you and forever grateful. Dad, the financial support you gave to me throughout the study will never be forgotten. I thank you mum for your encouragement when things were not working the way I wished to. Last but not least, to all my colleagues at the Plant Transformation Laboratory, Kenyatta University for their support during the study; Immaculate Mueni Mwangangi, Eric kuria, Joel Masanga, Sylvia Mbula Mutinda, Paul Karanja and Joshua Muli. May God bless you. v TABLE OF CONTENTS DECLARATION .................................................................................................... ii DEDICATION ....................................................................................................... iii ACKNOWLEDGMENTS ..................................................................................... iv TABLE OF CONTENTS ........................................................................................ v LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ............................................................................................... ix LIST OF APPENDICES ........................................................................................ xi ABBREVIATION AND ACRONYMS ............................................................... xii ABSTRACT ......................................................................................................... xiv CHAPTER ONE ..................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1 Background of the study ................................................................................... 1 1.2 Problem Statement ............................................................................................ 5 1.3 Justification ....................................................................................................... 6 1.4 Null hypotheses ................................................................................................. 8 1.5 Objectives ........................................................................................................ 8 1.5.1 General objective........................................................................................ 8 1.5.2 Specific objective ....................................................................................... 8 CHARPTER TWO.................................................................................................. 9 LITERATURE REVIEW ....................................................................................... 9 2.1 Origin, classification, distribution and infestation of Striga ............................. 9 2.2 Striga biology.................................................................................................. 11 2.2.1 Striga Germination ................................................................................... 11 2.2.2 Haustorium development ......................................................................... 12 2.2.3 Establishment of Striga parasitism and completion of the life cycle ....... 13 vi 2.3 Economic importance of Striga weeds and constrains of sorghum production due to Striga infestation ................................................................................ 15 2.4 Domestication and taxonomic classification of Sorghum bicolor .................. 16 2.6 Control measures to Striga infestation in SSA ............................................... 19 2.7.1 Low germination stimulant production .................................................... 22 2.7.2 Low production of the haustorial initiation .............................................. 23 2.7.3 Genetic basis for Striga resistance ........................................................... 23 2.8 Host plant cellular aspects against Striga parasitism ...................................... 25 2.9 Retrieving global arrays of gene expression in resistance .............................. 27 2.11 Functions of pathogenesis related proteins ................................................... 31 2.12 Wild sorghum as source of natural resistance to Striga parasitism .............. 32 CHAPTER THREE .............................................................................................. 34 MATERIALS AND METHODS .......................................................................... 34 3.1 Plant materials ................................................................................................. 34 3.1.1 Study area ................................................................................................. 35 3.1.2 Experimental design ................................................................................. 35 3.1.3 Germination of Striga seed ...................................................................... 35 3.1.4 Germination and growing of sorghum plants in rhizotrons ..................... 36 3.1.6 Quantification of post-attachment resistance ........................................... 37 3.1.7 Microscopic screening of Striga resistance .............................................. 37 3.2.1 Identification of Striga resistance genes from RNA-sequencing ............. 38 3.2.2 Quality control and processing of RNA-Seq data .................................... 38 3.2.3 Reference mapping of the reads to Sorghum bicolor genome ................. 39 3.2.4 Functional annotation ............................................................................... 40 3.2.5 Primer design............................................................................................ 40 3.2.6 RNA extraction and qualityfication ......................................................... 41 3.2.7 Complementary DNA synthesis (cDNA) and Polymerase Chain Reaction (PCR) ....................................................................................................... 42 vii 3.3 Statistical data analysis ................................................................................... 43 CHAPTER FOUR ................................................................................................. 44 RESULTS ............................................................................................................. 44 4.1 Assessments of Post-attachment Striga Resistance ........................................ 44 4.1.1 Germination of S. hermonthica Seeds ...................................................... 44 4.1.2 Growth of sorghum seedlings in Rhizotrons ............................................ 45 4.1.3 Macroscopic screening of Striga resistance ............................................. 45 4.1.4 Evaluation of resistance response of sorghum accession to Striga parasitism ................................................................................................. 47 4.1.5 Microscopic screening for Striga resistance ............................................ 52 4.2.1 Gene ontology .............................................................................................. 56 4.2.2 RT- PCR analysis of isolated PR genes ....................................................... 58 CHAPTER FIVE .................................................................................................. 59 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ...................... 59 5.1 Discussion ..................................................................................................... 59 5.1.1 Evaluation of wild sorghum accessions in response to Striga infestation 59 5.1.2 Expression of pathogenesis related proteins in wild sorghum ................. 63 5.2 Conclusion ...................................................................................................... 67 5.3 Recommendations ..........................................................................................