Evaluation of Flea Beetle (Phyllotreta spp.) Resistance in Spring and Winter-Type Canola (Brassica napus) by Julian R. Heath 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 © Julian R. Heath, September 2017 ABSTRACT EVALUATION OF FLEA BEETLE (PHYLLOTRETA SPP.) RESISTANCE IN SPRING AND WINTER-TYPE CANOLA (BRASSICA NAPUS L.) Julian R. Heath Co-Advisors: University of Guelph, 2017 Drs. Laima Kott and Istvan Rajcan This thesis is an investigation into the understanding of flea beetle (Phyllotreta spp.) resistance in spring-type and winter-type canola quality Brassica napus L.. Canola is one of the world’s most widely grown oilseed crops and an economically important crop in Western Canada. Genetic resistance to this common pest would add to the available tactics for integrated pest management of flea beetles in canola. The purpose of this research was to better understand the interactions of the flea beetle with canola at key feeding times in the life cycle of the flea beetle and identify genetic components related to flea beetle herbivory on canola seedlings. The objectives were to: 1) investigate seasonal effects of flea beetle herbivory on both spring-type and winter-type canola; and 2) identify quantitative trait loci (QTL) for flea beetle herbivory in two winter-type doubled haploid (DH) populations using simple sequence repeat (SSR) markers and single nucleotide polymorphic (SNP) markers. Year and seasonal effects were noted but overall trends amongst entries were similar concluding that flea beetle feeding patterns did not change throughout its life cycle. Spring-type and winter-type germplasm reacted similarly under flea beetle feeding. As such, there does not appear to be any novel resistance mechanisms that evolved as a result of divergent growth habit types in canola. Seven QTL were identified over the two DH populations studied. Linkage group (LG) N13 had multiple QTL identified. The remaining QTL were located on LG N04, N06, N15 and N17. It is unknown as to what mechanisms these QTL are associated with. The results of this thesis provide insight into flea beetle-canola plant interactions and identify some genetic areas of interest related to flea beetle herbivory in canola. ACKNOWLEDGEMENTS I would like to express my gratitude to all those who have supported me during this nine-year journey. Firstly, thank you to my co-advisors Dr. Istvan Rajcan and Laima Kott for your patience, support and encouragement throughout this process. This has been a challenging undertaking and without the support and strong encouraging words when needed, this may not have been successful. It has been a pleasure learning from you. I would like to thank my advisory committee of Drs. Ron Fletcher (deceased), Rebecca Hallett and Jay Patel. Sadly, Dr. Fletcher passed prior to seeing this completed but his insight with the material helped steer the project into what it became. Dr. Hallett provided a varying point of view coming from the insect world and provided fantastic feedback and direction throughout the process. Finally, thank you to Dr. Patel for whom I have had the pleasure of working with over the past 16 years. It was your guidance and support that got me started on this journey and helped me see it through to the end. Your guidance over the years for this research and beyond has been greatly appreciated. Thank you to the members of the examining committee for taking the time to review the manuscript and provide constructive feedback; Dr. Clarence Swanton (chair), Dr. Sally Vail (external examiner), Dr. Ali Navabi, Dr. Jay Patel and Dr. Istvan Rajcan. I wish to thank DuPont Pioneer for supporting me in this process. It has been a long journey and there have been many people that have assisted me over the years at various stages. A huge thank you to everyone that played a role – big or small, as without you, I would not have been able to do this. Special thanks to all my friends and family for understanding the time and commitment to work and school. Finally, thank you to my wife Chrissie and daughter Ellie for the love and support and tolerating me during the highs and lows of this journey. iv LIST OF ABBREVIATIONS AAFC Agriculture and Agri-Food Canada ANOVA Analysis of variance B Brassica Bt Bacillus thuringiensis CRISPR Cluster regularly interspaced short palindromic repeats C.V. Coefficient of variation DH Doubled haploid EPN Entomopathogenic nematodes F-Value F test statistics FBSC Flea beetle score GWAS Genome wide association study IST Insecticidal seed treatment LG Linkage group(s) LOD Logarithm/Likelihood of odds QTL Quantitative trait loci (locus) RCBD Randomized complete block design R² Coefficient of determination SD Standard deviation SE Standard error SNP Single nucleotide polymorphism SSR Simple sequence repeats TALEN Transcription activator-like effector nuclease ZFN Zinc finger nuclease v TABLE OF CONTENTS Abstract .............................................................................................................. ii Acknowledgements ........................................................................................... iv List of Tables ...................................................................................................... ix List of Figures .................................................................................................... xii List of Figures (Appendix) ................................................................................ xvii General Introduction .......................................................................................... 1 Chapter 1 - Literature Review ............................................................................. 6 1.1. The life history and biology of Phyllotreta cruciferae and P. striolata ........................................7 1.1.1. Description and life history of Phyllotreta species – P. cruciferae and P. striolata .............. 7 1.1.2. Flea beetle management ...................................................................................................... 9 1.2. Host plant selection and behavior of Phyllotreta spp. on Brassicaceae .................................... 14 1.2.1. Host plant selection ............................................................................................................ 15 1.2.2. Host plant acceptance ........................................................................................................ 19 1.2.3. Chemical cues ..................................................................................................................... 23 1.2.4. Physical cues ....................................................................................................................... 27 1.2.5. Cost of resistance ................................................................................................................ 30 1.3. Flea beetle (Phyllotreta spp.) responses to host plant secondary metabolites and morphology 31 1.3.1. Glucosinolates ..................................................................................................................... 31 1.3.2. Cyanogenic glycosides ........................................................................................................ 37 1.3.3. Alkaloids .............................................................................................................................. 38 1.3.4. Terpenoids .......................................................................................................................... 38 1.3.5. Phenolics ............................................................................................................................. 40 1.3.6. Other plant hormones, amino acids, proteinase inhibitors, etc. ....................................... 41 vi 1.3.7. Herbivore mechanisms of handling secondary metabolites .............................................. 43 1.3.8. Herbivore mechanisms of handling morphological defense .............................................. 44 1.4. Identifying the most promising plant chemical or physical feature to investigate in the development of flea beetle resistance in canola. ........................................................................... 45 1.4.1. Current works of interest .................................................................................................... 46 1.4.2. Transgenic or molecular-based resistance ......................................................................... 47 1.5. Summary ............................................................................................................................... 49 1.6. Hypothesis............................................................................................................................. 51 Chapter 2 - Evaluation of Winter-type Brassica napus Germplasm for Genetic Diversity in Response to Flea Beetle Herbivory in Typical and Atypical Planting Windows .......................................................................................................... 55 2.1. Abstract ................................................................................................................................. 55 2.2. Introduction .......................................................................................................................... 56 2.3. Materials and Methods .........................................................................................................