MARKER ASSISTED BREEDING TO TRANSFER STRIGA RESISTANCE IN SORGHUM FROM A MAPPED DONOR SOURCE N13 TO A FARMER PREFERRED KENYAN VARIETY OCHUTI JACKYLINE ABIGAIL. W. NGUGI Bsc. Biotechnology A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A DEGREE OF MASTER OF SCIENCE IN GENETICS AND PLANT BREEDING DEPARTMENT OF PLANT SCIENCE AND CROP PROTECTION FACULTY OF AGRICULTURE, UNIVERSITY OF NAIROBI, 2014 DECLARATION I declare that this thesis is my original work and has not been presented for a degree in any other University. Signature..……… …… Date……………………….. Jackyline Abigail .W. Ngugi This thesis is submitted to the Board of Postgraduate Studies with the approval of the following supervisors Signature …………………………………. Date…………………………. Dr. Kahiu Ngugi Department of Plant Science and Crop Protection University of Nairobi Signature ……………………………... Date………………………………. Prof. Eunice Mutitu Department of Plant Science and Crop Protection University of Nairobi Signature ……………………………... Date ………………………… Dr. Dan Kiambi Executive Director African Biodiversity Conservation and Innovations Centre Nairobi i DEDICATION This work is dedicated to my loved ones for their support and encouragement. ii ACKNOWLEDGEMENT I thank my supervisors: Dr. Kahiu Ngugi, Prof. Mutitu both of University of Nairobi, Dr. Dan Kiambi of African Biodiversity Conservation and Innovations Centre (ABCIC) and Dr. Santie de Villiers of International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) for their supervision and guidance. My sincere thanks and gratitude also go to Agrobiodiversity and Biotechnology Programme of the Association for Strengthening Agricultural Research in Eastern and Central Africa (ASARECA), particularly Dr. Charles Mugoya and Clet Wandui for the funding and support. I also thank the entire staff of International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) both in Kenya and India, staff for Biosciences of Eastern and Central Africa (BecA), International Livestock Research Institute (ILRI) and my collegues in the laboratory seven, classmates, technician Macharia and Carol and all those who made this study possible. Last but not least, my sincere gratitude goes to my husband, parents, siblings and everyone who gave me tremendous encouragement and hope especially when I had problems with my eyes. God bless you all. iii Table of Contents DECLARATION……………………………………………………………………………………..I DEDICATION ......................................................................................................................................II ACKNOWLEDGEMENT ................................................................................................................. III ABBREVIATIONS ........................................................................................................................... VII LIST OF TABLES .......................................................................................................................... VIII LIST OF FIGURE.............................................................................................................................. IX LIST OF PLATES................................................................................................................................ X LIST OF APPENDIXES...................................................................................................................... X ABSTRACT ........................................................................................................................................ XI CHAPTER 1 GENERAL INTRODUCTION 1.0 INTRODUCTION ............................................................................................................................... 1 1.1 SORGHUM PRODUCTION .........................................................................................................2 1.2 STRIGA............................................................................................................................................3 1.2.1 FLOWERS FOR DIFFERENT STRIGA SPECIES ................................................................................... 4 1.2.2 STRIGA IN KENYA......................................................................................................................... 6 1.2.3 STRIGA BIOLOGY.......................................................................................................................... 6 1.3 MARKER ASSISTED BREEDING ..............................................................................................8 1.4 PROBLEM STATEMENT AND JUSTIFICATION ................................................................10 1.5 OBJECTIVES................................................................................................................................10 1.5.1 GENERAL OBJECTIVE .................................................................................................................. 10 1.5.2 SPECIFIC OBJECTIVES.................................................................................................................. 10 1.6 HYPOTHESIS ...............................................................................................................................10 CHAPTER 2 LITERATURE REVIEW 2.1 SORGHUM ....................................................................................................................................11 2.1.1 SORGHUM REPRODUCTIVE BIOLOGY.......................................................................................... 12 2.1.2 CHARACTERISTICS OF SORGHUM VARIETIES IN KENYA .............................................................. 13 2.2 STRIGA..........................................................................................................................................14 2.2.1 STRIGA GERMINATION STIMULANTS ........................................................................................... 15 iv 2.2.2 STRIGA BIOLOGY........................................................................................................................ 16 2.2.3 MECHANISMS OF STRIGA RESISTANCE ....................................................................................... 18 2.2. 3. 1 Low Germination Stimulant (LGS) production................................................................ 19 2.2.3.2 Low Production of the Haustorial Initiation Factor (LHF) ................................................ 19 2.2.3.3 Hypersensitive Response (HR) ........................................................................................... 20 2.2.3.4 Incompatible Response (IR) ............................................................................................... 20 2.2.3.5 Mechanical barriers ............................................................................................................ 20 2.2.3.6 Antibiosis ............................................................................................................................ 21 2.3 GENETIC MARKERS .................................................................................................................21 2.3.1 HYBRIDIZATION-BASED MOLECULAR MARKERS ......................................................................... 22 2.3.1.1 Restriction fragment length polymorphism (RFLP) ........................................................... 22 2.3.2 PCR-BASED MARKERS................................................................................................................ 22 2.3.2.1 Random amplified polymorphic DNA (RAPD) ................................................................. 23 2.3.2.2 Amplified fragment length polymorphism (AFLP)............................................................ 23 2.3.2.3 Cleaved amplified polymorphic sequence (CAPS) ............................................................ 23 2.3.2.4 Sequence characterized amplified region (SCAR) ............................................................. 24 2.3.2.5 Sequence Tagged Site (STS) .............................................................................................. 24 2.3.2.6 Single Nucleotide Polymorphism (SNP) ............................................................................ 24 2.3.2.7 Simple Sequence Repeats (SSR) ........................................................................................ 24 2.3.2.8 Diversity Array Technology (DArT) .................................................................................. 25 2.4 MARKER ASSISTED SELECTION (MAS) .............................................................................25 2.4.1 FACTORS AFFECTING MAS......................................................................................................... 26 2.4.2 FOREGROUND AND BACKGROUND SELECTION ............................................................................ 26 2.5 SORGHUM MAPPING................................................................................................................28 2.5.1 STRIGA QTL MAPPING ......................................................................................................... 29 CHAPTER 3 BACKCROSS GENERATION AND STRIGA QTL ANALYSIS IN THE BACKCROSSES 3.1 INTRODUCTION .........................................................................................................................33 3.2 PLANT MATERIALS FOR GENERATION OF BACKCROSSES ........................................... 34 3.2. 1 GREEN HOUSE ACTIVITIES............................................................................................ 34 3.3 MATERIALS AND METHODS..................................................................................................35 3.4 FOREGROUND ANALYSIS.......................................................................................................36 3.4.1 DNA EXTRACTION ..................................................................................................................... 36 3.4.2 DNA QUALITY AND QUANTITY CHECK ....................................................................................... 37 3.4.3 FOREGROUND SELECTION ........................................................................................................... 38 3.4.4 POLYMERASE CHAIN REACTION (PCR)...................................................................................... 40 v 3.4.5 GENOTYPING .............................................................................................................................. 40 3.4.6 DATA ANALYSIS ......................................................................................................................... 42 3.5 RESULTS.......................................................................................................................................42 3.5.1 DNA QUALITY CHECK. ............................................................................................................... 42 3.5.2 FOREGROUND SELECTION ........................................................................................................... 45 3.6 DISCUSSION ................................................................................................................................46 3.7 CONCLUSION..............................................................................................................................49 CHAPTER 4 EVALUATION OF STRIGA RESISTANCE IN BACKCROSSES WITH INTROGRESSED RESISTANCE QTL IN WESTERN KENYA 4.1 INTRODUCTION .........................................................................................................................50 4.2 MATERIALS AND METHODS..................................................................................................51 4.2.1 FIELD TRIALS AT ALUPE AND KIBOS ............................................................................... 51 4.2.2 DATA COLLECTION AND STATISTICAL ANALYSIS ...................................................... 52 4.3 RESULTS.......................................................................................................................................54 4.3.1 EVALUATION OF STRIGA RESISTANCE IN KIBOS DURING MAY TO OCTOBER 2010 SEASON .................. 54 4.3.2 EVALUATION OF STRIGA RESISTANCE IN ALUPE DURING MAY TO OCTOBER 2010 SEASON ................. 57 4.3.3 EVALUATION OF STRIGA RESISTANCE IN KIBOS DURING OCTOBER 2010 TO MARCH 2011SEASON....... 59 4.3.4 EVALUATION OF STRIGA RESISTANCE IN ALUPE DURING OCTOBER 2010 TO MARCH 2011SEASON ...... 61 4.3.5 COMBINED ANALYSIS OF VARIANCE (ANOVA) ............................................................................... 63 4.3.5.1 MAY TO OCTOBER 2010 SEASON.................................................................................................. 63 4.3.5.2 OCTOBER 2010 TO MARCH 2011 SEASON ...................................................................................... 64 4.3.6 PHENOTYPIC CORRELATIONS RESULTS ............................................................................................ 66 4.3.6.1 Correlation for evaluation of Striga resistance in Kibos during May to October 2010 season ........ 66 4.3.6.2 Correlation for evaluation of Striga resistance in Alupe during May to October 2010 season ........ 68 4.3.6.3 Correlations for evaluation of Striga resistance in Kibos during October to March 2011 season .. 70 4.3.6.4 Correlations for evaluation of Striga resistance in Alupe during October to March 2011 season.... 72 4.3.7 AUSPC TOTAL ........................................................................................................................ 74 4.4 DISCUSSION............................................................................................................................... 75 4.5 CONCLUSION ............................................................................................................................ 78 CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1 CONCLUSION..............................................................................................................................79 5.2 RECOMMENDATIONS ..............................................................................................................80 6.0 REFERENCES ..............................................................................................................................81 7.0 APPENDIXES………………………………………………………………………………….105 vi ABBREVIATIONS ASARECA: The Association for Strengthening Agricultural Research in Eastern and Central Africa AUSPC: Area under Striga progressive curve ASNPC: Area Under the above ground Striga Number Progress Curve AUDPC: Area under the disease progress curve BC: Backcross BMZ: Federal Ministry for Economic Cooperation and Development (BMZ) cM: Centimorgan CTAB: Cetyl trimethyl-ammonium bromide DNA: Deoxyribonucleic acid ICRISAT: International Crops Research Institute for the Semi-Arid Tropics MAS: Marker Assisted Selection MABC: Marker Assisted Backcrossing PCR: Polymerase Chain Reaction QTL: Quantitative Trait Loci RNA: Ribonucleic acid RP: Recurrent parent SSR: Simple Sequence Repeat vii LIST OF TABLES Table 1. Comparison of sorghum nutritional value to other food crops……………………...……1 Table 2. Estimated area under Striga infestation in East and Central Africa…………………….5 Table 3. Summary of SSR markers used in the study ……………………………………….….....31 Table 4. Number of samples genotyped per generation ………………………………….………..35 Table 5. Details of the SSR markers used for foreground selection…………………………….....39 Table 6. Co-loading sets used in the study…………………………………………………..............41 Table 7. Results for foreground selection for BC F and BC3F2 (BC S )……………………..….46 3 1 3 1 Table 8. The reaction of 8 backcross generations to Striga at Kibos during May 2010 to October 2010………………………………………………………………………………………….…56 Table 9. The reaction of 8 back cross generations to Striga at Alupe during May 2010 to October 2010…………………………………………………………………………………………….58 Table 10. The reaction of 8 back cross generations to Striga at Kibos during October 2010 to March 2011…………………………………………………………………………………....60 Table 11. The reaction of 8 backcross generations to Striga at Alupe during October 2010 to March 2011………………………………………………………………….………………...62 Table 12. Genotype x Environment reactions of 8 back-cross generations in two locations during May 2010 to October 2010 and October 2010 to March 2011………….………………….65 Table 13. The correlation summary of 8back cross generations reaction to Striga at Kibos during May 2010 to October 2010……………………………………………..……....……67 Table 14. The correlation summary of 8 back cross generations reaction to Striga at Alupe during May 2010 to October 2010………………………………………………………..…69 Table 15. The correlation summary of 8 back cross generations reaction to Striga at Kibos during October 2010 to March 2011…………………………………………………….….71 Table 16. The correlation summary of 8 back cross generations reaction to Striga during October 2010 to March 2011………………………………………………………………..73 viii LIST OF FIGURE Figure 1. Sorghum yields in leading sorghum producing countries…………………………...…...2 Figure 2. Striga infestation Intensity in Africa…………………………………………...……….....6 Figure 3. Structures of Striga germination stimulants ………………………………...……….….16 Figure 4. Summary of Striga lifecycle …………………………………..……………….……….…18 Figure 5. Genetic linkage map of RIP (N13 X E36-1) ………………………………………...…..30 Figure 6. Summary of the foreground SSR markers used……………………………..……….…31 Figure 7. 0.8% Agarose gel showing the quality of genomic DNA from BC F …. ……………....43 3 1 Figure 8. A pie chart showing Nanodrop readings of 72 samples of BC F ……………………...44 3 1 Figure 9. A pie chart showing Nanodrop readings of 288 samples of BC F …………………......44 4 1 Figure 10 . Agarose gel image of amplified markers used in foreground selection………………………………………………………………………...……….…45 Figure 11. AUSPC total for the genotypes under Striga infestation for May 2010 to March 2010……………………………………………………………………………………....…74 ix