Winter-Hardy Spring Wheat Breeding: Analysis of Winter x Spring Wheat Germplasm and the Development of Selection Tools by Roger James Andrew Larsen A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctorate of Philosophy in Plant Agriculture Guelph, Ontario, Canada © Roger James Andrew Larsen, August, 2012 ABSTRACT Winter-Hardy Spring Wheat Breeding: Analysis of Winter x Spring Wheat Germplasm and the Development of Selection Tools Roger James Andrew Larsen Advisor: University of Guelph, 2012 Dr. Duane E. Falk Development of a winter-hardy spring wheat breeding platform could increase the gain in selection per year over traditional winter wheat breeding programs. To make use of spring wheat being able to produce three generations per year, an indoor cold tolerance screen using chlorophyll fluorescence (Fv/Fm) and visual assessment two weeks after freezing as evaluation parameters was developed. Evaluation of Ontario-adapted winter and spring wheat varieties demonstrated that the test was able to differentiate between winter and spring wheat. Specific varieties from this data set were used to develop an indoor freezing survival index (IFSI) to normalize data for effective ranking of germplasm in further experiments. Indepth analysis of a Froid (winter) x Siete Cerros (spring) wheat population using molecular markers indicated that a significant level of cold tolerance is preserved when the Vrn- B1 spring allele is used compared to the Vrn-A1 allele. Generation means analysis of the same cross indicated that the cold tolerance was due to additive genetic effects. Multiple populations with at least one spring parent were advanced to the F3:4 generation. IFSI analysis indicated that several lines from the populations had cold tolerance similar to Ontario-adapted winter wheats and better than several winter barley varieites. Further testing found a significant 5% improvement in cold tolerance was noted for spring wheat varieties treated with Cruiser Maxx seed treatment. Finally, a Norstar (winter) x Bergen (spring) doubled haploid wheat population was analysed and a significant correlation to LT50 data from an independent laboratory validated the methods used in these experiments. In a separate experiment, multiple indicies calculated from spectral reflectance measurements taken on the Ontario winter wheat performance trial at Elora and Harriston in 2008-09 were found to be significantly correlated to winter survival ratings. Fall reflectance measurements indicate non-random plant density or vigour effects in the trials. To adjust winter survival ratings accordingly, linear and non-linear approaches were used and found the non-linear model to be statistically superior. Large differences between locations illustrated that for complete modelling of winter survival, more data from locations of differing soil types, plant density and plant growth stage is required. ACKNOWLEDGEMENTS I would like to sincerely thank my advisor Dr. Duane Falk for mentoring me over the course of my studies. The main reason why I continued on in school was to hone my skills as a practical and classical plant breeder. His consistent encouragement to take a hands-on approach helped me to learn to be self-sufficient and gain a greater understanding of what it takes to be a cereals breeder. Our scientific, philosophical and personal conversations at school, in the field and during travel will always be remembered, and I am glad to say I enjoyed my time as a graduate student. His encouragement to get involved with committees inside and outside of the department led to recognition from SeCan as ‘Seed of the Year’. Furthermore, it led to collaborative research opportunities with private industry and the chance to become involved with the OCCC. All of these aspects were important for a young plant breeders education. Equally, I would like to thank Mark Etienne and Henry Olechowski at Hyland Seeds, my NSERC Industrial post-Graduate Scholarship and MITACS partners for being willing to fund my education and research. Mark is an excellent breeder. He is always open to new ideas, always willing to be patient, share advice with me, and always willing to include me as a member of his team. He could have viewed me as extra labour or bother; instead, he trusted me to make selections in the nursery, solicited me to provide advice in the breeding program and included me on breeding trips. His doubled haploid breeding program was different than the breeding program at Guelph, giving me knowledge and experience with another method of breeding. Mark’s rigourous and meticulous note taking helped me to understand the importance to be detailed in my approaches and his ability to pull light brown chaff out of a plot still amazes me! Henry was a constant source of support as a member of my advisory committee and as an industrial partner. He furthered my education by always including me in the business aspects of the iv Hyland cereals program, as well as, ongoing research projects in the corn and soybean programs. He was always willing to support my research interests by any means possible, which was not easy considering the constraints of a medium-sized, family owned breeding company. I would like to thank the two other members of my advisory committee, Dr. Hugh Earl and Dr. Laima Kott. Hugh was instrumental in encouraging me to work with chlorophyll fluorimetry and spectral reflectance to measure cold tolerance and winter survival. Laima was always available for me, willing to share ideas and even growth cabinet space for my experiments. Her lab is a source of constant innovation and it is something that I hope to replicate in my career. I would like to thank the members of the cereal breeding lab, Zorka Zslavnics and the gang. They were a great help in planting, managing plots, and harvesting. They were also a source of good conversation and practical knowledge. I would like to thank the staff members of the Department of Plant Agriculture. Specifically, I would like to thank Peter Smith, Dietmar Scholz and Jim Hoare for all of their assistance. We had a strong cohort of graduate students while I was in the department and I will fondly remember dating myself at every coffee break. I will also remember the sharing of ideas and co-learning which is really what school is all about. Finally, I would like to acknowledge my family for being a constant source of support. My wife Angela deserves most of the recognition for tolerating me going back to school, listening to my scientific drivel, having two amazing children while I was in school, working to support me and doing with less and never complaining. v TABLE OF CONTENTS ABSTRACT II ACKNOWLEDGEMENTS IV LIST OF TABLES XI LIST OF FIGURES XVI LIST OF ABREVIATIONS XIX INTRODUCTION 1 CHAPTER 1. LITERATURE REVIEW 4 1.2 The Evolution of Wheat (Triticum sp.) 4 1.2.1 Pre-Domestication Evolution 4 1.2.2 Domestication and Cultivation of Wheat 9 1.3.1 Wheat Production in the World 12 1.3.2 Wheat Production in Canada 14 1.3.3 Wheat Production in Ontario 17 1.4 Mechanisms for Control of Flowering in Wheat 19 1.4.1 Vernalization 19 1.4.2 Photoperiod 28 1.4.3 The Flowering Pathway in Cereals 32 1.4.4 Gene specific Molecular Markers for Vernlization and Photoperiod 33 1.5 Cold Tolerance in Cereals 35 1.5.1 Winter Survival in Ontario 35 1.5.2 Effect of Cold on Plant Cells 35 1.5.3 Effects of Low Temperatures on Photosynthesis 36 1.5.4 Effect of Reactive oxygen species on the Photosystems 36 1.5.5 Cold Temperature Effects on Plant Cell Membranes 37 1.6 Plant Cell Responses to Cold and Mechanisms to Avoid Cold Temperature Induced Damage 37 1.6.1 Alteration of Plant Cell Membranes and Membrane Composition 38 vi 1.6.2 Alterations to the Plant Cell Wall 38 1.6.3 Change in Plant Metabolite Concentrations 39 1.6.4 Alteration of Solute Concentrations 39 1.6.5 Alteration in Light Energy Usage and Photosynthetic Pathways 40 1.6.6 Genetic Mechanisms of Cold tolerance and Activation of Genes involved in Acclimation to Cold Temperatures 41 1.7 Methods for Testing Cold Tolerance in Cereals 44 1.7.1 Electrolyte Leakage 45 1.7.2 Chlorophyll Fluorescence 46 1.7.2.1 THE BIPARTITE MODEL 46 1.7.2.2 PHOTOSYNTHESIS (PQ) 46 1.7.2.3 NON-PHOTOCHEMICAL QUENCHING (NPQ) 47 1.7.2.4 CHLOROPHYLL FLUORESCENCE (FQ) 47 1.7.2.5 FLUORESCENCE MEASURES OF DARK ADAPTED LEAVES (MAXIMUN QUANTUM YIELD OF PHOTOSYSTEM II) 48 1.7.3 Application of Fv/Fm for Testing Cold Stress in Cereals 49 1.7.4 Assessment of Cold Tolerance Outdoors 51 1.8 Using Spectroradiometry for Evaluating Biomass in Field Crops 52 1.8.1 The Development of Spectral Reflectance as a Tool to Assess Plant Leaves 52 1.8.2 Factors Affecting Canopy Reflectance 53 1.8.3 Informative Reflectance Ratios 54 1.8.3.1 NORMALIZED DIFFERENCE VEGETATIVE INDEX 54 1.8.3.2 SOIL ADJUSTED VEGETATIVE INDEX 55 1.8.3.3 TRIANGULAR VEGETATIVE INDEX 56 1.8.4 Canopy Reflectance as a Measure of Biomass 56 CHAPTER 2. DEVELOPING THE WINTER HARDY SPRING WHEAT PLATFORM 75 2.1 Introduction 75 2.2 Materials and Methods 82 2.2.1 Experiment #1- Testing of Ontario-adapted spring and winter wheat varieties 82 2.2.1.1 GERMPLASM 82 2.2.1.2 EXPERIMENTAL DESIGN, PLANT GROWTH CONDITIONS 82 2.2.1.3 CHLOROPHYLL FLUORESCENCE MEASUREMENTS 83 2.2.1.4 VISUAL ASSESSMENT OF WHEAT PLANTS TWO WEEKS POST FREEZING 84 2.2.1.5 FIELD EVALUATION FOR ONTARIO-ADAPTED WINTER WHEAT 84 2.2.1.6 STATISTICAL ANALYSIS 85 2.2.1.7 CALCULATION OF LT50 85 2.2.1.8 INDOOR FREEZING SURVIVAL INDEX (IFSI) OF ONTARIO ADAPTED WINTER AND SPRING