The Genetic Architecture of Spawning Date and the Associations Among Life History Traits and Growth in Rainbow Trout (Oncorhynchus mykiss) By Melissa S. Allen A Thesis presented to The University of Guelph In partial fulfilment of requirements For the degree of Doctor of Philosophy In Integrative Biology Guelph, Ontario, Canada © Melissa S. Allen, June, 2015 ABSTRACT THE GENETIC ARCHITECTURE OF SPAWNING DATE AND THE ASSOCIATIONS AMONG LIFE HISTORY TRAITS AND GROWTH IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) Melissa Allen Advisors: University of Guelph, 2015 Professor R. G. Danzmann Professor M. M. Ferguson I investigated the genetic architecture of spawning date through quantitative trait locus (QTL) and candidate gene analyses and correlations among life history traits in a commercial hatchery strain of rainbow trout that has been under strong selection for spawning date. I first tested whether differentiation at the population level was occurring between early and late spawning broodstock at genetic loci throughout the rainbow trout genome. I detected significant genetic heterogeneity at microsatellite loci between females with early and late spawning dates within a season and this genetic differentiation was pronounced enough to assign females to the correct spawning group with an average accuracy of 76%. Many loci exhibiting significant differences in allele frequencies co-localize to genomic regions containing QTL for spawning date and other life history traits and potential candidate genes related to circadian rhythms and the brain-pituitary-gonadal axis (BPG axis). I next tested for associations among life history and growth traits by examining whether selection for spawning date based on the genetic markers associated with this trait results in differential growth, embryonic developmental rate and age of maturation. Families produced through selection of genetic markers associated with late spawning had significantly faster developmental rates and increased precocious male maturation. Co-localization of the two QTL detected for developmental rate to the same markers known to be associated with spawning date in this strain suggests that some of the co-variation between spawning date and developmental rate has a genetic basis. Furthermore, faster developmental rate conferred a growth advantage up to 13 months post fertilization within families and body size was a significant predictor of the propensity to mature early both within and across families. Finally, I tested whether variation in 9 candidate genes belonging to the clock gene system and BPG axis is associated with variation in spawning date. I detected 255 SNPs and 45 INDEL’s within the coding and non-coding regions of candidate genes. SNPs from three genes belonging to the clock gene system (bmal, clock1b, dec2) showed nominally significant associations with spawning date, providing further evidence that circadian genes play an important role in the circannual rhythms of salmonids. ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisors Dr. Roy Danzmann and Dr. Moira Ferguson for their support and encouragement during the course of my PhD. Their guidance has been invaluable during this process and I am truly fortunate to have had this opportunity. I would also like to thank my committee members Dr. Glen Van Der Kraak and Dr. Lewis Lukens for their guidance, suggestions and constructive criticism over the years. My lab members in Guelph have made this a more enriching experience, so a thank you to Xia Yue, Anne Easton, Marcia Chaisson, Andrea Kockmarek, Eva Kuttner, and Joe Norman for their advice and support. Moreover, your friendship has meant a great deal to me. More thanks to Aaron Goldt, Riley Magee, Colin and Cameron Richardson. A special thanks to my family for their unwavering support throughout my life. Words cannot express how grateful I am to my parents Sharon and Doug Allen, for all of the sacrifices they have made on my behalf. I would not be where I am today without their continual guidance and encouragement to strive towards my goals. Last, but definitely not least, I would like to thank my husband, Leonard, for always being there for me for better and for worse. His faith in me has been a driving force in my life and I will be forever grateful for his patience and support. iv TABLE OF CONTENTS Acknowledgements ……………………………………………………………………………... iv List of Tables ...…………………………………………………………………………………. vi List of Figures …………………………………………………………………………………. viii Chapter I: General Introduction ……………………………………………………………….. 1 Chapter II: Molecular markers for variation in spawning date in a hatchery population of rainbow trout (Oncorhynchus mykiss) …………………………………………………………. 12 Introduction …………………………………………………………………………………….. 13 Material and Methods ..………………………………………………………………………… 16 Results ………………………………………………………………………………………….. 20 Discussion ……………………………………………………………………………………… 23 Chapter III: Marker assisted selection for spawning date and co-variation among economically important fitness traits in a commercial strain of rainbow trout (Oncorhynchus mykiss) ……... 38 Introduction .……………………………………………………………………………………. 39 Material and Methods ………………………………………………………………………….. 43 Results ………………………………………………………………………………………….. 48 Discussion ……………………………………………………………………………………… 51 Chapter IV: Candidate gene sequencing reveals variants in circadian clock genes associated with spawning date in rainbow trout (Oncorhynchus mykiss) …………………………………. 76 Introduction……………………………………………………………………………………...77 Material and Methods……………………………………………………………………………80 Results ………………………………………………………………………………………….. 83 Discussion ……………………………………………………………………………………… 87 Chapter V: Conclusions and Future Directions …………………………………………….. 111 Appendices …………………………………………………………………………………… 114 Appendix A: Additional Files for Chapter II …………………………………………………. 114 Appendix B: Additional Files for Chapter IV ………………………………………………… 120 v List of Tables Table 2.1 Sixty three marker loci distributed among all 29 linkage groups used for genotype analysis of females in the LYNDON broodstock of rainbow trout. References are given for the 15 linkage groups known to contain spawning date QTL………………………………………34 Table 2.2 The linkage group and associated microsatellite markers displaying significant differences in allelic distributions between early and late spawning groups of female rainbow trout. Markers are grouped according to the significance thresholds of p< 0.001, 0.01 and p< 0.05 following sequential Bonferroni testing (Rice 1989). For each marker, the alleles whose frequency differed significantly between early and late spawning groups are given in the last column. The BELS ranking for each marker is also provided, where l indicates the marker with the highest discriminatory power……………………………………………………………….35 Table 3.1 Microsatellite markers used in the selection index to choose male rainbow trout with genotypes associated with earlier and later spawning dates. The alleles showing significant differences in frequencies between early spawning females (mid-August to mid-October) and late spawning females (mid-November to early January) are given for each locus, along with their direction of effect (negative indicates an allele associated with early spawning, positive indicates an allele associated with late spawning)………………………………………………63 Table 3.2 Mean hatching time (± standard error) of progeny from four sets of maternal half-sib families (1 female crossed to multiple males). The vector score and corresponding predicted spawning date genotype of each male parent is given. Significant differences in mean hatching time (hours since the first embryo hatched) within maternal half-sib families are denoted by lower case letters, where shared letters indicate no significant difference in mean time to hatch (p< 0.01). Significant differences in mean hatching time between maternal half-sib groupings are denoted by upper case letters, where shared letters indicate no significant difference in mean time to hatch (p< 0.01)………………………………………………………………………….64 Table 3.3 Mean length (± standard error) at the onset of exogenous feeding (males and females combined) in the progeny of a female mated to four male rainbow trout with either an early spawning vector score (E) or late spawning vector score (L) . The mean body weights (BW) (grams) at 13 and 20 months of age (from fertilization) by sex from the early, middle and late hatching groups for the four maternal half-sib families are also given. Significant differences in trait values (p< 0.05) between hatching groups are denoted by lower case letters, where shared letters indicate no significant difference. Significant differences in trait values (p< 0.05) between half-sib families are denoted by upper case letters, where shared letters indicate no significant difference………………………………………………………………………………………..65 Table 3.4 The mean condition factor, K, (± standard error) at 13 and 20 months of age (from fertilization) by sex from the early, middle and late hatching groups for the progeny of a female mated to four male rainbow trout with either an early spawning vector score (E) or late spawning vector score (L). Significant differences in trait values (p< 0.05) between hatching groups are denoted by lower case letters, where shared letters indicate no significant difference. Significant vi differences in trait values (p< 0.05) between half-sib families are denoted by upper case letters, where shared letters indicate no significant difference…………………………………………67 Table 3.5 The number and percentage of