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Utilization of Alien Genetic Diversity for Improving Drought Tolerance in Bread Wheat

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Utilization of Alien Genetic Diversity for Improving Drought Tolerance in Bread Wheat Utilization of Alien Genetic Diversity for Improving Drought Tolerance in Bread Wheat By Maria Khalid (Registration No: NUST201390130TPASAB8013F) Thesis Supervisor: Dr. Alvina Gul Department of Plant Biotechnology Atta-ur-Rahman School of Applied Biosciences National University of Sciences & Technology (NUST) Islamabad, Pakistan (2019) Utilization of Alien Genetic Diversity for Improving Drought Tolerance in Bread Wheat By Maria Khalid (Registration No: NUST201390130TPASAB8013F) A thesis submitted to the National University of Sciences and Technology, Islamabad, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Applied Biosciences Thesis Supervisor: Dr. Alvina Gul Co-Supervisor: Dr. Rabia Amir Department of Plant Biotechnology Atta-ur-Rahman School of Applied Biosciences National University of Sciences & Technology (NUST) Islamabad, Pakistan (2019) Dedication I dedicate all my research work to my father Mr. Abdul Rashid Khalid and my mother Mrs. Nasreen Akhtar for the understanding and encouragement they provided during this course of study. They have supported me all the way since the beginning of my studies. They have never failed to give me financial and moral support, providing for all of my needs during this time and for teaching me that even the biggest task can be accomplished if it is done one step at a time. i Acknowledgment First of all, I am thankful to Almighty Allah for His blessings, guidance, and knowledge that He has bestowed upon me. I am really thankful to Him that He with His mercy helped me face every challenge that I faced during the entire course of my studies and all through my life. Praise Be to Him alone, The owner of honor and majesty. I am really thankful to Almighty Allah who made me amongst the followers of His last and final messenger and prophet Muhammad (SAW). His (SAW) teaching have helped me to come up with every problem I have ever plunged into during the course of studies and during my life as a whole. I would like to express my deepest gratitude to my respected supervisor Dr. Alvina Gul who has been a tremendous mentor to me. I have been extremely lucky to have a supervisor like her who has always been very responsive to my queries in her sweet and kind way. She provides her students with a very comfortable working environment. She is not only a good supervisor but a good person by heart. I am deeply indebted to her for her patient guidance and motivation throughout my thesis. I would like to extend my special thanks to Dr. Awais Rasheed for his support, patience, and guidance. I am tremendously fortunate to work under his supervision and guidance throughout my Ph.D. journey. He is an exceptional scientist and a great human being. At many stages in the course of this thesis, I benefited from his advice, particularly so when exploring new ideas. His positive outlook and confidence in my research inspired me and gave me confidence. I would also want to thank my co-supervisor; Dr. Rabia Amir for her support and the examination committee members; Dr. Muhammad Faraz Bhatti, Dr. Faiza Munir and Dr. Sumaira Farrakh for their valuable feedback and support. They spent their precious time to answer my queries. I am extremely thankful to them. I would also like to mention my foreign supervisors Dr. Zhonghu He (Institute of Crop Science, National Wheat Improvement Center, Chinese Academy of Agricultural Sciences, CAAS), Dr. Hikmet Budak (Molecular Biology, Genetics & Bioengineering. Sabanci University, Istanbul-Turkey) and Dr. Lee Hickey (Queensland Alliance for Agriculture and Food innovation, University of Queensland, Brisbane, ii Australia) for hosting me as a visiting researcher in their labs and providing a great platform for my PhD research. Without their support and guidance, it would have been impossible to complete my research. To my parents for their love and affection who always acted as a moral support for me. Without their interest and cooperation, I would not have ever been able to carry out this task. Thank you for your love, support and unwavering belief in me. Without you, I would not be the person I am today. I would like to thank my sister and best friend Dr. Zoya Khalid for her love and concern for me. She has always encouraged me to go abroad and gain more exposure and helped me in my research as well. She always boosted my mood whenever I felt down. I would have never completed my task without her support. Thank you so much for always being there for me. Above all, I would like to thank my husband Zubair Ahmad whose unconditional love, patience and support for my academic career enabled me to complete my thesis. I could not have reached my goals without his support. Thank you so much for putting up with me through the toughest time of my life. I am thankful to Allah for enlightening my life with your presence. I am thankful to my amazing institute National University of Sciences and Technology for providing me a platform to do my Ph.D. I am thankful to the Higher Education Commission (HEC) for providing me IRSIP scholarship to complete my Ph.D. research in Australia. I am thankful to TUBITAK scholarship program for providing me an opportunity to work in Turkey. I am also thankful to Food Security Scholarship (FSC) program to provide me short-term scholarship and make me able to gain experience by doing research work in China. In last, I would like to thank all my friends, who have been a great source of support for me, my Ph.D. colleagues with whom I have shared moments of concern and all my teachers who taught and encouraged me Maria Khalid iii Table of Contents S. No. Title Page No. i Dedication i ii Acknowledgment ii ii List of abbreviations vi iii List of figures x iv List of tables xii v Abstract xiii 1. INTRODUCTION & LITERATURE REVIEW 1 1.2 Origin and cultivation of wheat 2 1.1. Origin of einkorn, emmer, dinkel and kamut 3 1.3. Hybridization of BBAADD component of wheat 4 1.4. Stresses of wheat 6 1.5. Drought stress in wheat 6 1.5.1. Effect of drought stress at seedling stage 7 1.5.2. Effect of drought stress at flowering stage 8 1.5.3. Effect of drought stress at seed developing stage 8 1.6. Drought tolerance in wheat 9 1.6.1. Genes conferring drought tolerance in wheat 9 1.6.1.1. Role of cell wall invertase gene in drought 10 1.6.2. Breeding wheat for drought tolerance 11 1.7. Role of CIMMYT in wheat production 11 1.7.1. International Triticeae Mapping Initiative 12 1.7.2. Synthetic hexaploid wheat 16 1.7.2.1. History and production of synthetic hexaploid wheat derived 16 population 1.7.2.2. Synthetics by bread wheat derivatives (SH/BW) 18 1.7.3. The contribution of synthetic hexaploid wheat in drought tolerance 18 1.8. Measures of genetic diversity 19 1.9. Non-neutral markers/Functional markers 20 1.10. Genetic mapping and drought tolerance 26 1.10.1. Genetic linkage map construction 28 1.10.2. QTL mapping 29 1.11. Aims and objectives 31 2. MATERIALS & METHODS 32 2.1. Mutations in wheat cell wall invertase gene (TaCwi-B1) underpin 32 drought tolerance in synthetic/bread wheat derivatives 2.1.1. Germplasm collection of synthetic derivatives 32 2.1.2. Phenotyping of synthetic derivatives under glasshouse conditions 32 2.1.3. Phenotyping of synthetic derivatives under field conditions 33 2.1.4. DNA extraction of synthetic derivatives 34 2.1.5. Primer designing and PCR amplification of synthetic derivatives 35 iv 2.1.6. Gel electrophoreses and gel elution from PCR product 36 2.1.7. Cloning of TaCwi-B1 38 2.1.8. Nucleotide sequence retrieval and alignment of TaCwi-B1 39 2.1.9. Analysis of DNA indels with novoSNP of TaCwi-B1 39 2.1.10. Protein sequence retrieval and alignment of TaCwi-B1 39 2.1.11. Protein Variation Effect Analyzer (PROVEAN) of TaCwi-B1 39 2.1.12. Protein structure prediction of TaCwi-B1 40 2.1.13. Prediction of disordered regions of TaCwi-B1 40 2.1.14. Prediction of protein stability changes of TaCwi-B1 40 2.1.15. Analyzing point mutations by Hope server in TaCwi-B1 41 2.1.16 Phylogenetic analysis of TaCwi-B1 41 2.2. Genome wide linkage mapping for seedling morphology under 41 drought stress in ITMI (w7984)/Opata 2.2.1. Germplasm collection and phenotyping of ITMI population 41 through cigar roll method 2.2.2. Genotyping by sequencing of ITMI population 43 2.2.3. Statistical analysis of phenotypic and genotypic data of ITMI 46 population 2.2.4. Linkage map construction and linkage analysis 46 2.3. Allelic effects of major genes controlling developmental traits and 48 drought tolerance in a diversity panel derived from synthetic wheat derivatives 2.3.1. Genotyping of SYN-DER by using KASP assays 48 2.3.2. Development of KASP assays for TaSST genes 49 2.3.3. Statistical analysis by using Tassel software 50 3. RESULTS 51 3.1. Mutations in cell wall invertase gene (TaCwi-B1) underpin drought 51 tolerance in derived synthetic wheat 3.1.1. Allelic variation in TaCwi-A1, TaCwi-B1 and TaCwi-D1 and their 52 effects on phenotypes 3.1.2. Analysis of DNA indels in TaCwi-B1 with novoSNP 52 3.1.3. Mutation analysis by PROVEAN of a TaCwi-B1 52 3.1.4. Protein structure prediction of TaCwi-B1 53 3.1.5.
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