HOMOEOLOGOUS RECOMBINATION-BASED CHROMOSOME ENGINEERING FOR PHYSICAL MAPPING AND INTROGRESSION IN WHEAT AND ITS RELATIVES AEGILOPS SPELTOIDES AND THINOPYRUM ELONGATUM A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Mingyi Zhang In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Program: Genomics and Bioinformatics January 2020 Fargo, North Dakota North Dakota State University Graduate School Title HOMOEOLOGOUS RECOMBINATION-BASED CHROMOSOME ENGINEERING FOR PHYSICAL MAPPING AND INTROGRESSION IN WHEAT AND ITS RELATIVES AEGILOPS SPELTOIDES AND THINOPYRUM ELONGATUM By Mingyi Zhang The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Xiwen Cai Chair Phillip McClean Steven S. Xu Justin D. Faris Zhaohui Liu Approved: January 22, 2020 Phillip McClean Date Department Chair ABSTRACT Wheat (genome AABBDD) is one of the essential crops, offering approximate 20% of human calorie consumption worldwide. Allopolyploidization of three diploid ancestors led to hexaploid wheat with narrowed genetic variation. Chromosome engineering is an applicable approach to restore the evolutionarily-omitted genetic diversity by homoeologous chromosomes recombination between wheat and its relatives. Two diploid relatives of wheat, Thinopyrum elongatum (genome EE) and Aegilops speltoides (genome SS), containing favorable genes, are used as gene resources for alien introgression and genome diversification in wheat. An advanced and effective experiment procedure was developed and applied for the production, recovery, detection, and characterization of homoeologous recombinants. Meanwhile, a novel recombinant chromosome recovery strategy was exploited with improved efficiency and accuracy. In this study, recombinants of wheat chromosomes 3B and 7B with their homoeologous chromosomes in Th. elongatum and Ae. speltoides (i.e. 3B-3E, 7B-7E, and 7B-7S) were produced and detected. Totolly, 81 3B-3E recombinants and four aberrations involving in distinct chromosomal regions were developed in three recombination cycles by fluorescent genomic in situ hybridization (FGISH). The secondary and tertiary recombination breakpoints occurred toward the proximal regions comparing to the primary recombination under this advanced recombination procedure. A novel recovery strategy was used to recover 7B-7E and 7B-7S homoeologous recombinants by chromosome-specific markers and FGISH verification. Marker-based pre-screening and subsequent FGISH verification identified 29 7B-7E and 61 7B- 7S recombinants, seven 7B-7E and four 7B-7S Robertsonian translocations, one 7E and five 7S telocentric chromosomes, and three 7S deletions. All the recombinants and aberrations were genotyped by high-throughput wheat 90K single nucleotide polymorphism (SNP) assay and the iii recombination breakpoints were physically mapped to wheat chromosome 3B or 7B according to their FGISH patterns, SNP results, and wheat reference genome sequence. Chromosome 3B was physically partitioned into 38 bins with 429 SNPs. Meanwhile, 44 distinct bins were resolved for chromosome 7B with 523 SNPs. A composite bin map was constructed for chromosomes 3B and 7B, respectively, with a comprehensive analysis of FGISH and SNPs results. In summary, this project provides a unique physical framework for further wheat genome studies and diversifies the wheat genome for germplasm development in wheat breeding. iv ACKNOWLEDGEMENTS I would thank everyone who has supported and assisted me during my whole doctoral study. First and foremost, I would like to express my deepest gratitude to my supervisor, Dr. Xiwen Cai, whose wealth of knowledge, research attitude, and his encouragement and patience make a great impact on my life and guide me through my doctoral program. I would sincerely appreciate my graduate committee members, Drs. Phillip McClean, Steven S. Xu, Justin D. Faris, and Zhaohui Liu, who have supported my research goals and improved my professional studies. I would like to thank Dr. Shiaoman Chao, who has helped me on wheat 90K SNP genotyping and GenomeStudio operating. I also express my sincere thanks to our lab technician, Wei Zhang, who helped me on the course’s study and experiment execution. I extend my grateful thanks for my previous and current lab members, Rachel McArthur, Xianwen Zhu, Shaungfeng Ren, Tatiana Danilova, Yadav Gyawali, and Somo Ibrahim for their friendship and collaboration. I would also express my gratitude to the Program of Genomics and Bioinformatics, the Department of Plant Sciences, North Dakota State University (NDSU) for providing me this opportunity to pursue my Ph.D. Nobody has been more important than my family members during my Ph.D. studies. I would thank my parents and brother for their support and inspiration. I wish to thank my husband, Qing Sun, and my daughter, Chloe Sun, to provide endless love and happiness for me. v TABLE OF CONTENTS ABSTRACT...................................................................................................................................iii ACKNOWLEDGEMENTS ............................................................................................................ v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x CHAPTER 1. GENERAL INTRODUCTION ............................................................................... 1 References ................................................................................................................................... 3 CHAPTER 2. LITERATURE REVIEW ........................................................................................ 7 Wheat origin, evolution, and domestication ................................................................................ 7 Taxonomy of wheat ................................................................................................................. 7 Origin and evolutionary of polyploid wheat ........................................................................... 9 Domestication of wheat ......................................................................................................... 10 Wheat genome and chromosomes ............................................................................................. 11 Wheat genome ....................................................................................................................... 11 Wheat chromosomes ............................................................................................................. 13 Chromosome engineering-based gene introgression in wheat .................................................. 14 Wheat gene pools .................................................................................................................. 14 Chromosome engineering in wheat ....................................................................................... 15 Gene introgression in wheat and its relatives ........................................................................ 16 Meiotic homoeologous recombination-based alien introgression ......................................... 17 References ................................................................................................................................. 19 CHAPTER 3. PARTITIONING AND PHYSICAL MAPPING OF WHEAT CHROMOSOME 3B AND ITS HOMOEOLOGUE 3E IN THINOPYRUM ELONGATUM BY INDUCING HOMOEOLOGOUS RECOMBINATION .............................. 30 Abstract ..................................................................................................................................... 30 Introduction ............................................................................................................................... 31 vi Material and methods ................................................................................................................ 34 Plant materials ....................................................................................................................... 34 Homoeologous recombination population development ....................................................... 34 Molecular cytogenetic analysis ............................................................................................. 34 Molecular marker analyses .................................................................................................... 36 Results ....................................................................................................................................... 36 Induction and recovery of 3B-3E recombinants and aberrations .......................................... 36 Detection and delineation of 3B-3E recombinants and aberrations by FGISH ..................... 38 FGISH-based physical analysis of 3B-3E recombination breakpoints ................................. 43 Homoeologous recombination-based physical mapping of wheat chromosome 3B ............. 44 Development of chromosome 3E-specific STARP markers and construction of a comparative physical map for chromosomes 3E, 3B, 3A, and 3D........................................ 47 Discussion ................................................................................................................................