Genotypic and Phenotypic Analysis of a Nepali Spring Wheat (Triticum aestivum L.) Population by Kamal Khadka A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Plant Agriculture Guelph, Ontario, Canada © Kamal Khadka, May, 2020 ABSTRACT GENOTYPIC AND PHENOTYPIC ANALYSIS OF A NEPALI SPRING WHEAT (TRITICUM AESTIVUM L.) POPULATION Kamal Khadka Advisor(s): Dr. Alireza Navabi University of Guelph, 2020 Dr. Manish N. Raizada Nepal has been completely dependent on introduced wheat (Triticum aestivum L.) germplasm for variety development despite having >500 landraces in the national genebank. No Nepali wheat genetic resources were involved in the development of any of the 43 varieties released in Nepal for commercial cultivation. Nepal’s capacity to genotype and phenotype its wheat germplasm, in order to utilize it for breeding, is in its infancy due to a lack of resources. To assist breeding efforts for Nepal, here, I hypothesized that: (1) Nepali spring wheat germplasm is genetically and phenotypically diverse; (2) that the important physio- morphological traits have a genetic basis; and (3) that promising accessions for future targeted breeding can be identified using such genotyping and phenotyping. I assembled the Nepali Wheat Diversity Panel (NWDP) consisting of 318 spring wheat accessions including landraces, CIMMYT lines and released varieties. The NWDP was phenotyped in four different field experiments (2 each in Nepal and Canada) and also under controlled conditions. Analysis of 95K high density GBS markers showed greater genetic diversity in the Nepali landrace group compared to modern germplasm. Unexpectedly, the population structure analysis revealed four, rather than 3 subpopulations as was originally expected based on breeding history, with significant admixture within each subpopulation. A genome-wide association study (GWAS) revealed 15 significant marker-trait associations (MTAs) for 6 agro-morphological traits. Targeted genotyping was conducted to assess the accessions for allelic variation at dwarfing loci (Rht) and a photoperiod insensitivity locus (Ppd), both targets of modern selection. Promising accessions for future breeding were identified that possessed dwarfing alleles but conversely also seedling vigour related traits with potential to promote early season drought tolerance. The NWDP also showed significant variation for NDVI, SPAD values and shoot waxiness. I suggest that the Nepali landraces should be further characterized to identify the “authentic” landraces while the genotypic information available should be further utilized in genomic selection. The data suggest that shoot waxiness may be confounding spectral reflectance measurements especially when a germplasm population is extremely diverse. In conclusion, it is hoped that this thesis will better inform and accelerate wheat breeding for Nepal. Keywords: Nepali Wheat Diversity Panel, population structure, GWAS, physio-morphological traits, dwarfing alleles, NDVI, waxiness DEDICATION This thesis is dedicated to Dr. Alireza Navabi, a one-of-a-kind professor, great mentor, dedicated breeder and friend. Thank you for the trust that you had in me. iv ACKNOWLEDGEMENTS First of all, I would like to extend my heartfelt gratitude to my primary advisor, Dr. Alireza Navabi, who passed away in early 2019 fighting bravely with pancreatic cancer; for his thorough and continuous support until the last few days of his life. Dr. Navabi was an excellent mentor, a friend, and a great human being. I feel proud that I had the opportunity to work with such a great soul. I would like to express my sincere thanks to Dr. Manish N. Raizada for his support throughout these five years not just as a co-advisor but more like a guardian. I am extremely grateful to Dr. Raizada’s endless support and guidance from the day I met him which has in fact, influenced my career. In addition, I would also like to candidly thank Professors Hugh Earl and Zeny Feng for serving on my Ph.D. advisory committee and advising me on my project to improve the quality of research and writing. In particular, Dr. Earl was very supportive despite his extremely busy schedule. Also, I thank Dr. Andrew J. Burt for helping me out on my research writing after the passing away of Dr Navabi. Further, Dr. Davoud Torkamaneh and Dr. Mina Kaviani deserve genuine acknowledgement for their support in handling genotypic data and manuscript edits. Special thanks go to Harwinder Singh Sidhu and Dr. Mitra Serazajari for their constant support and encouragement throughout these five years. I feel lucky that I had the opportunity to work with a bunch of great colleagues in the Wheat Breeding Lab who helped me enormously to take care of my research work. I would like to thank the lab technicians, Nick Wilker, Melinda Drummond, and Kat Bosnic, for their great support in managing seed and field trials in Canada. Also, helping hands from Ryan Costello, Zhang Zhanghao, Kaitlyn Ranft and Elijah Dalton were highly valued. I cannot stop thanking my other lab members Emily Gordon, Andy Chen, Dr. Soren Seifi and Dr. Ismael Schegoscheski, for their support and beautiful friendship. I also thank the members of Raizada lab especially Dr. Malinda Thilakarathna, Dr. Tejendra Chapagain, Dr. Travis Goron and Finlay Small for their support and friendship. v Furthermore, the growth facility coordinators in the Crop Science Building, Dietmar Scholz and Sue Couling, deserve appreciation from me. I would also like to recognize the primary funding from the Canadian International Food Security Research Fund (CIFSRF), jointly funded by the International Development Research Centre (IDRC, Ottawa) and Global Affairs Canada, and partial funding from SeCan, the Agricultural Adaptation Council and Grain Farmers of Ontario. In addition, the collaborator for Nepal field experiment Nepal Agricultural Research Council (NARC, Nepal) is highly acknowledged. Finally, I have to thank my wife, Anuja, and my daughter, Aarohi, for their love, support and patience through good and bad times during these five years. At this moment, I would like to remember my parents, brother and sister in Nepal, who had a big dream for me, for their love and patience to finally achieve this. vi TABLE OF CONTENTS Abstract .......................................................................................................................................... ii Dedication ..................................................................................................................................... iv Acknowledgements ....................................................................................................................... v Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xv List of Figures ............................................................................................................................ xvii List of Symbols, Abbreviations and nomenclature ................................................................. xix List of Appendices ...................................................................................................................... xxi 1 General Overview ................................................................................................................. 1 2 Review of the Literature ....................................................................................................... 3 2.1 Abstract ............................................................................................................................ 3 2.2 Wheat in the context of global food security ................................................................... 3 2.3 Status of wheat research in Nepal .................................................................................... 4 2.4 Factors affecting wheat productivity ................................................................................ 7 2.4.1 Key biotic and abiotic factors affecting wheat yield ................................................ 7 2.4.2 Factors affecting wheat productivity in Nepal .......................................................... 8 2.5 Breeding high yielding, stress tolerant wheat varieties .................................................... 9 2.5.1 The physiological approach to wheat breeding ...................................................... 10 2.5.2 Molecular approaches to wheat breeding ............................................................... 11 2.5.3 Genome-wide association mapping (GWAS) ......................................................... 12 2.5.4 Kompetitive Allele-Specific PCR (KASP) ............................................................. 12 2.5.5 Genetic diversity for breeding better wheat varieties ............................................. 13 2.6 Physio-morphological traits for breeding high yielding wheat varieties ....................... 14 vii 2.6.1 Phenological traits ................................................................................................... 14 2.6.2 Coleoptile length ..................................................................................................... 14 2.6.3 Chlorophyll content ...............................................................................................