Gene expression analyses help elucidate positive selection in cultivated crops and early seedling responses to thiamethoxam and neighbouring turf grass by Megan Alexandra House 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 © Megan Alexandra House, January, 2016 ABSTRACT GENE EXPRESSION ANALYSES HELP ELUCIDATE POSITIVE SELECTION IN CULTIVATED CROPS & EARLY SEEDLING RESPONSES TO THIAMETHOXAM AND NEIGHBOURING TURF GRASS Megan Alexandra House Advisor: University of Guelph, 2015 Associate Professor Lewis N. Lukens This thesis investigates the role of gene expression in crop evolution and crop responses to environmental variation. It applies both retrospective and experimental approaches to examine how changes in gene expression contribute to plant improvement. Using a retrospective approach, I identified genes whose regulatory alleles were selected during the breeding of elite rice varieties. Selection for groups of genes with shared attributes likely altered oxidative stress responses and amylase activity. I also identified 31 genes that have strong-acting trans regulatory factors and may have been selected for increased or decreased expression in rice breeding. In maize, I then used a high-throughput sequencing approach to identify candidate genes from seedlings that have a transcriptional response to experimentally manipulated weed competition (turf grass). Weed competition altered transcript abundance of 102 genes. Gene function annotation indicates that genes related to carbon metabolism, ROS-scavenging, hormone signalling and defence are involved in the early response of maize to the weed treatment. With the wide-use of neonicotinoid insecticides, I was also interested if thiamethoxam, a component of a popular insecticide used for maize, has an effect on seedling response to weeds. I tested for an interaction between seedling response to weeds and the thiamethoxam treatment, and found that thiamethoxam affects the response of genes involved in photosynthesis, defense, and hormone signalling. I believe that candidate genes from both approaches will be useful for future breeding efforts. Acknowledgements I would first like to thank my advisor, Dr. Lewis Lukens, for giving me the opportunity to become the researcher that I am today. My successes as a graduate student would not have been possible without him. I would also like to thank Dr. Cortland Griswold for supporting me as a member of my committee, and for his assistance with my first publication and his revisions to this thesis. Thank you as well to the rest of my advisory committee, Dr. Elizabeth Lee and Dr. Steven Rothstein, for your help in the completion of my thesis and for your support along the way. I am also very grateful to Dr. Clarence Swanton for allowing me to join his weed team, and for being a constant source of encouragement. Thank you to all of my labmates who have helped me along the way. A very special thank you to Dr. Ann Meyer and Dr. Greg Downs for all their assistance with everything related to computers and for all of their moral support and encouragement. I literally would not have been able to do this without them. I would also like to thank my family and friends for all of their support throughout the years. Livia and Sarah, we share everything, and your endless support throughout the past 6 years has helped me more than you know. Thank you to my mom and dad who have been there for me no matter what I needed - food, money, laughter. It has not gone unappreciated. Thank you to my sister for providing moral support and for producing the cutest child ever born to distract me when days were difficult. iv Last, but of course not least, I would like to thank my partner Gerald who has been there for me no matter what the circumstances. He has talked science with me, come to the school on the weekends with me, supported and encouraged me, taken care of me, and has believed in me. v Table of Contents ABSTRACT ......................................................................................................... iv Acknowledgements ........................................................................................... iv List of Tables ...................................................................................................... ix List of Figures ..................................................................................................... x List of Abbreviations ......................................................................................... xi CHAPTER 1 Introduction ................................................................................ 1 CHAPTER 2 Literature Review ....................................................................... 3 2.1 PROCESSES OF ARTIFICIAL SELECTION .......................................................... 3 2.1.1 Origins of plant domestication ......................................................... 3 2.1.2 The influence of modern plant breeding .......................................... 5 2.2 GENOMIC SIGNATURES OF SELECTION ........................................................... 6 2.2.1 Selection acts on protein-coding DNA, epialleles, and DNA encoding regulatory regions .......................................................................................... 6 2.2.2 Approaches for identifying the molecular effects of selection .......... 9 2.2.3 A novel retrospective approach to identifying candidates for future breeding 14 2.2.4 Limitations of the test of independence for inferring selection ....... 18 2.3 MORPHOLOGICAL AND PHYSIOLOGICAL RESPONSES TO THE ENVIRONMENT ...... 19 2.3.1 The morphological and physiological responses of plants to low R:FR growth conditions ............................................................................... 19 2.3.2 Thiamethoxam alters the morphological and physiological response of maize seedlings to neighbouring turf grass ............................................. 22 2.4 HYPOTHESES AND OBJECTIVES: .................................................................. 26 CHAPTER 3 Evidence for selection on gene expression in cultivated rice (Oryza sativa)… ................................................................................................. 28 vi 3.1 ABSTRACT ................................................................................................. 29 3.2 INTRODUCTION ........................................................................................... 30 3.3 MATERIALS AND METHODS .......................................................................... 33 3.3.1 Data Sources ................................................................................. 33 3.3.2 Detecting lineage-specific selection on functionally related genes and the genes' attributes.............................................................................. 34 3.3.3 Investigating lineage-specific selection on gene expression through reinforcing eQTL .......................................................................................... 36 3.4 RESULTS ................................................................................................... 40 3.4.1 Genes involved in shared processes have evidence of selection .. 40 3.4.2 Positive selection likely acted upon strong effect cis and trans eQTL during rice breeding ..................................................................................... 41 3.5 DISCUSSION ............................................................................................... 54 CHAPTER 4 Analysis of the early transcriptional response to neighbouring turf grass, and the effect of thiamethoxam on this response, in maize………. .................................................................................................. 62 4.1 ABSTRACT ................................................................................................. 62 4.2 INTRODUCTION ........................................................................................... 64 4.2.1 The presence of neighbouring plants induces changes in morphology and physiology ......................................................................... 64 4.2.2 Physiological changes associated with light quality: carbon metabolism, ROS-scavenging, hormone signaling and biotic defense ........ 65 4.2.3 Effect of thiamethoxam seed-treatment on the response to neighbouring weeds ..................................................................................... 67 4.3 MATERIALS AND METHODS .......................................................................... 69 4.3.1 Plant material and growth conditions ............................................. 69 4.3.2 RNA Isolation ................................................................................ 72 4.3.3 Processing and mapping of Illumina 100 bp paired-end reads ...... 72 4.3.4 Identification of differentially expressed genes .............................. 73 vii 4.3.5 Gene ontology enrichment analysis using AgriGO and PageMan . 75 4.4 RESULTS AND DISCUSSION ................................................................. 78 4.4.1 RNA-Seq identifies up- and down-regulation of genes in response to the weed treatment, including a xyloglucan endotransglucosylase involved in cell expansion .......................................................................................... 78 4.4.2 Weed treatment alters expression of genes involved in the photosynthetic light