Integrating Genomics and Metabolomics to Inform Breeding for Powdery Mildew Resistance in Grapevine A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Soon Li Teh IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Adrian D. Hegeman & James J. Luby August 2018 © Soon Li Teh 2018 Acknowledgements Words are inadequate to express my utmost gratitude to my advisors, Professor Adrian D. Hegeman and Professor Jim J. Luby, for navigating me through my graduate education. I am indebted to their dedication and expertise in nurturing and ‘fathering’ me in the fields of metabolomics and genetics. Their gracious support allowed me to engage in collaborative research projects, participate in technical training, as well as travel to national and international conference meetings. It was a great privilege to be your student over the years! I would like to acknowledge my advisory committee members, Professors Robert Blanchette, Jim Bradeen and Matthew Clark for their advice and guidance in various aspects of this project, particularly in areas of my research blind spots. Special thanks to Dr. Matthew Clark, an excellent scientist and a good friend, for his continued mentoring, editorial feedback, and ready availability. Much of the work in this dissertation would not have been possible without the collaboration of Dr. Lance Cadle-Davidson (USDA-ARS), Professor Anne Fennell (SDSU), Dr. Jonathan Fresnedo-Ramírez (OSU), Dr. David Gadoury, Dr. Elizabeth Demmings, Bety Rostandy, and Mani Awale. Notably, Drs. Cadle- Davidson and Gadoury were gracious hosts who provided technical training on controlled fungal inoculation in Geneva, NY. My heartfelt thanks to John and Jennifer Thull for their viticultural support and ready availability. Chapters 2 and 4 would not have existed without them! Many thanks to the members of Cohen-Gardner-Hegeman and Clark-Hokanson- Luby groups for their feedback and helpful discussion. In particular, the support of Dr. Nicholas Howard, John Tillman and Luke Haggerty was tremendous. I owe my due respects to Dr. Alison Pawlus, Dr. Dana Freund and Dr. Arthur Eschenlauer for their selflessness in nurturing me in the areas of natural products, metabolomics, and statistical analysis, respectively. Their guidance helped me build a strong foundation in these areas. I am also grateful for a conducive environment to pursue a graduate study that was made possible by Professor Emily Hoover, who leads the Department of Horticultural Science, as well as Lynne Medgaarden and the Director of Graduate Studies, who coordinate the Applied Plant Sciences. Funding was largely provided by the University of Minnesota and the USDA-NIFA-SCRI. I am also appreciative of a number of awards that provided financial support and recognition, including the UMN Doctoral Dissertation Fellowship, William H. Alderman Graduate Award, and Daniels Fellowship in Horticulture. I extend my cordial thanks to friends and family for always believing in me. My mom, my dad and my sisters have been a constant source of encouragement. To Huey Yee Lim, whose love is unconditional, thank you for your unwavering support throughout this journey and beyond. Finally, I thank the Lord Almighty for the opportunity and privilege to pursue a path I dearly love. I am thankful for His constant love and faithfulness, especially when the path was arduous and gruesome. i Dedication This dissertation is dedicated to farmers, particularly those in impoverished nations. Your unfailing drive to provide for the families has been a source of inspiration and reflection. May this work and my future endeavors be one step closer to bridging the chasm of poverty and malnourishment. ii Abstract Two powdery mildew resistance loci have been identified using pedigree- connected F1 mapping families at the University of Minnesota grape breeding program. A consensus linkage map of the resistant parent (MN1264) was developed for genetic mapping. The resistance loci were mapped on chromosomes 2 and 15, with additive effects accounting for over 30% phenotypic variation. Marker haplotypes, hap+chr2 and hap+chr15, were constructed to trace the inheritance of resistance loci in grandparent-parent-progeny relationships. Both hap+chr2 and hap+chr15 in the resistant F1 progeny were inherited from parent MN1264, that originated from grandparent ‘Seyval blanc’. Additionally, two microsatellites markers (i.e., UDV-015b and VViv67) were identified to be associated with hap+chr15, and can be applied for marker-assisted selection. In a follow-up study to characterize metabolic changes attributed to hap+chr2 and hap+chr15, a metabolomic experiment was conducted on whole-plant propagated grapes in a time-course response to in vivo inoculation. The use of several multivariate analyses systematically identified 52 biomarkers that were associated with hap+chr2, and 12 biomarkers with hap+chr15. In a temporal assessment of biomarkers, the discriminating metabolic changes distinguishing resistant and susceptible individuals appeared to be occurring from 24 to 48 hours after inoculation. iii Table of Contents Page List of Tables ix List of Figures x Chapter 1 Literature Review .................................................................... 1 1.1 Introduction ..................................................................................... 1 1.2 Goal, Objectives and Significance .................................................. 2 1.3 The Host: Grapevine ...................................................................... 3 1.4 The Pathogen: Erysiphe necator .................................................... 5 1.5 Genetics ......................................................................................... 7 1.6 Defense Response in Grapevine .................................................... 9 1.7 Stilbene Phytoalexins: Metabolic Response against Powdery Mildew ............................................................................. 9 1.8 The Tools: Genomic and Metabolomic Resources ......................... 12 Figures ...................................................................................................... 15 Chapter 2 Genetic Dissection of Powdery Mildew Resistance in Interspecific Half-Sib Grapevine Families Using SNP-based Maps ..................................................................... 18 2.1 Introduction ..................................................................................... 19 2.2 Materials and Methods ................................................................... 22 2.2.1 Plant Material ............................................................................ 22 2.2.2 Quantification of Field Powdery Mildew Severity ....................... 23 2.2.3 DNA Extraction, Sequencing, and SNP Assignment ................. 24 iv 2.2.4 Linkage Maps ............................................................................ 25 2.2.5 Construction of a Consensus Linkage Map ............................... 26 2.2.6 QTL Analysis ............................................................................. 27 2.2.7 Haplotype Construction and Analysis with Ren3 SSR .............. 28 2.2.8 Haploblock Validation Using Controlled Inoculation .................. 29 2.3 Results ........................................................................................... 29 2.3.1 Genotyping: Sequencing and SNP Assignment ........................ 29 2.3.2 Parental and Consensus Linkage Maps .................................... 30 2.3.3 Field Assessment of Powdery Mildew Severity ......................... 31 2.3.4 Analysis of Marker-Trait Association ......................................... 31 2.3.5 Haplotypes Associated with Powdery Mildew Resistance ......... 32 2.3.6 Haploblock Validation Using Controlled Inoculation .................. 34 2.4 Discussion ...................................................................................... 35 2.4.1 Genotyping: Sequencing and SNP Assignment ........................ 35 2.4.2 Parental and Consensus Maps ................................................. 36 2.4.3 Field Assessment of Powdery Mildew Severity ......................... 38 2.4.4 QTL Detection and Validation ................................................... 38 2.4.5 Haplotypes Associated with Powdery Mildew Resistance ......... 41 2.4.6 Co-localization with Ren3? ........................................................ 42 2.4.7 Chromosome 2: Ren10 and Potential Structural Dynamics in the Vicinity ............................................................................. 43 2.5 Conclusions .................................................................................... 44 Tables ....................................................................................................... 46 v Figures ...................................................................................................... 52 Chapter 3 Genetic Analysis of Stilbenes in Grapevine Stems Indicates an mQTL Hotspot for Disease Resistance Motifs on Chromosome 18 ..................................................... 62 3.1 Introduction ..................................................................................... 63 3.2 Materials and Methods ................................................................... 65 3.2.1 Sampling of Plant Material ........................................................ 65 3.2.2 Sample Preparation and Extraction ........................................... 65 3.2.3 Metabolic Profiling