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Transcriptome Analysis and Snp Marker Identification of Finger Millet (Eleusine Coracana (L.) Gaertn) at Critical Stages of Striga (Striga Hermonthica) Infestation

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Transcriptome Analysis and Snp Marker Identification of Finger Millet (Eleusine Coracana (L.) Gaertn) at Critical Stages of Striga (Striga Hermonthica) Infestation TRANSCRIPTOME ANALYSIS AND SNP MARKER IDENTIFICATION OF FINGER MILLET (ELEUSINE CORACANA (L.) GAERTN) AT CRITICAL STAGES OF STRIGA (STRIGA HERMONTHICA) INFESTATION Mikwa Erick Owuor (BSc. Biology) I56/24662/2012 A Thesis Submitted in Partial Fulfilment 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 APRIL, 2019 ii DECLARATION I, Mikwa Erick Owuor, duly declare that the work presented in this thesis is my original work and has not been presented for a degree or any other award in any other university or any other institution Mikwa Erick Owuor (B.Sc. Biology) I56/24662/2012 Signature……………………………. Date………………………………… We hereby confirm that the candidate carried out the work reported in this thesis under our supervision Dr. Mark Wamalwa Signature…………………...Date………………………… Department of Biochemistry and Biotechnology, Kenyatta University Dr. Richard O. Oduor Signature……………...........Date………………………. Department of Biochemistry and Biotechnology, Kenyatta University Dr. Damaris A. Odeny Signature…………………...Date………………………… Genetic Gains, ICRISAT-ESA, Nairobi iii DEDICATION This thesis is dedicated to my mother Zilpah Mikwa, her sacrifice, belief, motivation and self-denial has led me this far. iv ACKNOWLEDGEMENTS I would like to thank God Almighty for His guidance in strength and health to the end my course. I would like to thank my supervisors Dr. Mark Wamalwa, Dr. Damaris Odeny and Dr. Richard Oduor for their support, encouragements and supervision throughout the course. I appreciate the effort and time they took towards correction and improving the clarity of the thesis. I am very grateful to Dr. Damaris Odeny specifically for believing in me and accepting me in the finger millet project at International Crop Research Institute for Semi-Arid Tropics (ICRISAT). Many thanks to Prof. Mathew Dida (Department of Botany and Horticulture, Maseno University) and Dr. Rasha Ali (Wad Medani Agricultural Research Station, Sudan) for providing the necessary plant materials I needed for the project. At Biosciences east and central Africa–International Livestock Research Institute (BecA-ILRI) Hub, I am grateful to Dr. Francesca Stomeo, Solomon Maina, Mercy Macharia, James Oguya and Joyce Njuguna for their support during my fellowship. At ICRISAT, I thank Samuel Manthi, Rajneesh Palwaal and Vincent Njunge for their support during my fellowship in their lab. I want to especially thank Dryland cereals group through CGIAR Research Program on Dryland Cereals (CRP-DC) for sponsoring part of my MSc. and part of my project. BecA-ILRI Hub under the Africa Bioscience Challenge Fund (ABCF) program and Bio-resources Innovations Network for Eastern Africa Development (Bio-innovate) for sponsoring the first part of my project, and making it part of the finger millet whole genome annotation project. v I am very grateful to my family, my mum, sisters Ruth and Emma, and brothers Fred and Zachary for their tremendous love and support. I further extend my gratitude to my fellow students and fellows like Davis, Sandra, Godfrey, Easter, Milcah, Umar, and Erick for their continued support and advice whenever I needed it. Thank you very much vi TABLE OF CONTENTS DECLARATION ..........................................................................................................................ii DEDICATION ............................................................................................................................. iii ACKNOWLEDGEMENTS ........................................................................................................ iv TABLE OF CONTENTS ............................................................................................................ vi LIST OF TABLES ...................................................................................................................... ix LIST OF FIGURES ..................................................................................................................... x LIST OF APPENDICES ............................................................................................................. xi ABBREVIATIONS AND ACRONYMS ................................................................................. xiii ABSTRACT ................................................................................................................................ xv CHAPTER ONE .......................................................................................................................... 1 GENERAL INTRODUCTION .................................................................................................. 1 1.1 Background information........................................................................................................... 1 1.2 Problem statement and Justification ......................................................................................... 6 1.3 Null Hypotheses ....................................................................................................................... 7 1.4 Objectives ................................................................................................................................. 8 1.4.1 General Objectives ...................................................................................................... 8 1.4.2 Specific Objectives ..................................................................................................... 8 1.5 Significance of the study .......................................................................................................... 8 CHAPTER TWO ......................................................................................................................... 9 LITERATURE REVIEW ........................................................................................................... 9 2.1 Finger millet origin, distribution and adaptation ...................................................................... 9 2.2 Global economic importance .................................................................................................. 10 2.3 Taxonomy and botany ............................................................................................................ 12 2.4 Genetic diversity .................................................................................................................... 13 2.5 Production constraints ............................................................................................................ 14 2.5.1 Striga weed on finger millet ...................................................................................... 15 2.5.2 Striga life cycle ......................................................................................................... 16 2.5.3 Striga resistance mechanisms ................................................................................... 18 2.5.4 Strigolactone biosynthetic pathways and pre-germination resistance ....................... 19 2.5.5 Gene for gene resistance and Striga virulence .......................................................... 21 2.5.6 Mechanisms of Striga resistance in Sorghum and maize .......................................... 22 vii 2.5.7 Control strategies ...................................................................................................... 22 2.5.8 Screening for Striga resistance breeding resources................................................... 23 2.6 Genomics-assisted breeding in finger millet .......................................................................... 25 2.6.1 Next Generation Sequencing..................................................................................... 27 2.6.2 Transcript analysis .................................................................................................... 28 2.6.3 Single nucleotide polymorphisms (SNPs) identification .......................................... 30 CHAPTER THREE .................................................................................................................. 32 MATERIALS AND METHODS .............................................................................................. 32 3.1 Agronomic competence of the finger millet accessions and in vitro response to Striga exposure ................................................................................................................................ 32 3.2 Infecting of pre-germinated finger millet seedlings with Striga ............................................ 33 3.3 RNA extraction ...................................................................................................................... 34 3.4 Library preparation and sequencing ....................................................................................... 35 3.5 Quality control, de novo assembly of the sequences, and abundance estimation .................. 36 3.6 Differential gene expression analysis ..................................................................................... 37 3.7 Gene prediction, annotation and classification ....................................................................... 37 3.8 SNP analysis ........................................................................................................................... 38 CHAPTER FOUR ..................................................................................................................... 39 RESULTS ..................................................................................................................................
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