Stephanie Pedersen. A Thesis presented to The University of Guelph

Transcription

1 The Mapping of Quantitative Trait Loci Associated with Morphometrics and Parr Marks in an F 2 cross of European and North American Strains of Cultured Atlantic Salmon by Stephanie Pedersen A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada Stephanie Pedersen, April, 2013

2 ABSTRACT THE MAPPING OF QUANTITATIVE TRAIT LOCI ASSOCIATED WITH MORPHOMETRICS AND PARR MARKS IN AN F 2 CROSS OF EUROPEAN AND NORTH AMERICAN STRAINS OF CULTURED ATLANTIC SALMON Stephanie L. Pedersen University of Guelph, 2013 Advisor: Dr. E. G. Boulding Mapping quantitative trait loci (QTL) for traits under consideration for genetic improvement is becoming more common for many aquatic species, including Atlantic salmon. The objective of the study was to map QTL associated with length, weight, shape, parr mark number and contrast in three F 2 hybrid families of European and North American strains of Atlantic salmon using single nucleotide polymorphisms. GridQTL software was used to perform separate analyses for male and female linkage maps. Numerous highly significant QTL were detected for every trait. Locations of QTL differed based on age and map used. Some QTL locations for the analyzed traits were similar to those of other studies on purebred and backcross Atlantic salmon populations; however, many more QTL were detected in the hybrid F 2 s. The amount of genetic variation in skin colour and pattern displayed within the transatlantic F 2 families greatly exceeded the ranges seen in nature.

3 iii Acknowledgments I ll begin by thanking my wonderful advisor Dr. Liz Boulding who has provided me with so many opportunities and wisdom throughout the years. Without you I wouldn t be where I am today. You re a constant source of encouragement and positivity that made this whole experience so exceptional. You took me in as a third year undergrad student who barely knew anything about research or lab work and have stuck with me ever since, I can never thank you enough. I would like to thank my advisory committee members, Dr. Roy Danzmann and Dr. Brian Glebe. Roy, you put up with me dropping by your office without any notice and were always willing to help, your expertise motivated me to expand my knowledge beyond the obvious. Brian, you taught me so much about the aquaculture industry and policy that added so much to my appreciation of the field. You were always eager to help out when needed, even if that meant long drives to the airport or a homemade barbeque feast. I need to give a huge thank you to Steve Leadbeater, without you my fish may not have made it past one day let alone two years. Thank you for putting up with constant phone calls for help and room access. I can t express my appreciation for all your assistance in New Brunswick, and while I was writing my thesis. To my work-study students Simon, Josie and Adrienne, I owe you all so much. You were each exceptional and always got your work done, even under ridiculous timelines. Thank you so much to Michael for the wonderful fish illustrations, Lei for the linkage map, Tom and Chrissy for helping me with the (not so fun) task of DNA extractions, Chrissy again for the help with stats and R, Jon for the focus, and Holly for the constant emotional support. Finally I want to thank my wonderful and encouraging family, friends and co-workers for always being there when I needed you. Even when I was so stressed you probably didn t want me around, you could never let me keep the smile away for long. I have developed and learned so much during my M.Sc. and it s all thanks to the support of those around me. Funding from NSERC Strategic to EGB.

4 iv Table of Contents Acknowledgments... iii List of Tables... vi List of Figures... vii List of Appendices... viii Introduction Atlantic Salmon in Aquaculture Atlantic Salmon Lifecycle Parr Marks and their Importance in Atlantic Salmon Conservation A Brief History of Selection in Livestock Species Introduction to Genetic Markers, QTL Mapping, and Linkage Mapping Marker Assisted Selection North American and European Atlantic salmon Subspecies Objective, Hypothesis and Predictions Materials and Methods Creation of F 2 trans-atlantic hybrid crosses General Husbandry SNP Development DNA Analysis Morphometric Data Linkage Map QTL Detection Results Phenotype statistics QTL detection, length Length QTL Female map Length QTL Male map Comparing length QTL detected with female and male maps QTL detection, weight Weight QTL Female map Weight QTL Male map Comparing weight QTL detected with female and male maps QTL detection, parr mark number and contrast Parr mark QTL Female map Parr mark QTL Male map Comparing parr mark QTL detected with female and male maps QTL detection, morphometric landmarks Morphometric QTL Female map Morphometric QTL Male map Comparing morphometric QTL detected with female and male maps Discussion Colour Related Traits... 24

5 v Qualitative look at colour variation An examination of the genetic components of colour variation Smolting in the F 2 s Length and Weight Length and weight in F 2 s in comparison to the literature Examination of candidate genes associated with length and weight Homeologous Chromosomes Morphometric Traits Accounting for Differences Between Families Conclusions and Implications References Tables Figures Appendices... 69

6 vi List of Tables Table 1 Description of landmark positions and x/y coordinate names Table 2 Summary of means for phenotypic traits in all families of F 2 transatlantic salmon progeny Table 3 Linkage groups associated with significant QTL for length and weight at first measurement period using map based on female meioses Table 4 Linkage groups associated with significant QTL for length and weight at first measurement period using map based on male meioses Table 5 Linkage groups associated with significant QTL for length and weight at second measurement period using map based on female meiosis Table 6 Linkage groups associated with significant QTL for length and weight at second measurement period using map based on male meioses Table 7 Linkage groups with significant QTL for parr mark number and relative contrast of skin and parr mark colouration using map based on female meioses Table 8 Linkage groups with significant QTL for parr mark number and relative contrast of skin and parr mark colouration using map based on male meioses Table 9 Linkage groups with significant QTL for 11 landmarks at first measurement using map based on female meioses Table 10 Linkage groups with significant QTL for 11 landmarks at first measurement period using map based on male meioses Table 11 Linkage groups with significant QTL for 11 landmarks at second measurement period using map based on female meioses Table 12 Linkage groups with significant QTL for 11 landmarks at second measurement period using map based on male meioses Table 13 QTL for weight as described by Reid et al. (2005), Boulding et al. (2008), and Gutierrez et al. (2012) in comparison to the present study... 57

7 vii List of Figures Figure 1 Atlantic salmon lifecycle Figure 2 Landmarks for morphometric digitizing with description of positions Figure 3 Landmark variation based on centroid size Figure 4 Visualization of measurement for parr mark/skin contrast Figure 5 Distribution of length at first measurement Figure 6 Distribution of weight at first measurement Figure 7 Distribution of length at second measurement Figure 8 Distribution of weight at second measurement Figure 9 Distribution of total number of parr marks per individual Figure 10 Distribution of relative contrast of background skin colouration to parr marks Figure 11 Examples of colour morphologies from second measurement period... 68

8 viii List of Appendices Appendix 1 Female-specific linkage map for all three F 2 transatlantic salmon families Appendix 2 Male-specific linkage map for all three F 2 transatlantic salmon families... 71

9 1.0 Introduction 1.1 Atlantic Salmon in Aquaculture The use of salmon in aquaculture is a relative recent development in the scope of agricultural practices. Atlantic salmon were first cultured in Norway during the 1960 s with first harvest in 1971 (Gjedrem et al., 1991). In Canada salmon farming began in the 1970 s (Agriculture and Agri-Food Canada, 2008). Due to the overall decline of wild anadromous Atlantic salmon stocks, the majority of the world s salmon biomass is a result of aquaculture (Parrish et al., 1998), with wild Atlantic salmon accounting for less than 1% of the species production for human use (FAO, 2012). In Canada, Atlantic salmon account for over 90% of the total finfish in aquaculture, which valued over $653 million in 2009 (Fisheries and Oceans Canada, 2011). 1.2 Atlantic Salmon Lifecycle Anadromous Atlantic salmon are characterized by five distinct life stages: alevin, fry, parr, smolt, and post-smolt (or adult) (Figure 1). Fish that have newly hatched, named alevins, reside in the gravel of freshwater streams where they feed on their yolk sac. After approximately 3-8 weeks, alevins emerge from the gravel and begin to feed independently as fry. Juvenile Atlantic salmon, termed parr remain in their freshwater habitats for 1-8 years. Once parr have reached a threshold size between 10-20cm in length and are exposed to proper seasonal cues, the fish undergo what is termed the parr-smolt transformation, wherein parr undergo major physiological and morphological changes allowing them to tolerate saltwater (Björnsson et al., 2012, Thorstad et al., 2011). The smolts then migrate out to sea where they feed in the open ocean for 1-5 years before returning to their native freshwater rivers in order to spawn (Thorstad et al., 2011). The lifecycle of salmon in aquaculture follows the same principles as those in the wild, however occurs in a considerably more condensed timeframe. During their juvenile stages, Atlantic salmon are reared in heated freshwater tanks for months before the majority show signs of undergoing the parr-smolt transformation. At this time all fish, regardless of having smoltified, are transferred to large saltwater pens where they remain, maturing after an additional 2-4 years (Aksnes et al., 1986, Jonsson and Jonsson, 2006). 1

10 1.3 Parr Marks and their Importance in Atlantic Salmon Conservation Salmon parr develop dark oval markings along their lateral sides called parr marks (Behnke and Tomelleri, 2002), which vary in number and colour intensity across individuals and populations. It has been shown that the number of parr marks per individual has a large genetic component (Kudo et al., 2002); however environmental factors also play a role (Mezzera et al., 1997). Multiple hypotheses exist for the purpose of parr marks, with Maeda and Hidaka (1979) noting that the Japanese trout (Oncorhynchus masou) frequently flash their parr marks as an aggressive display in maintaining territory. The most widely accepted hypothesis, however, is that these areas of dark colouration are utilized for crypsis (Donnelly and Dill, 1983). In Atlantic salmon, studies have provided strong evidence to indicate that parr marks are an anti-predator adaptation to stream environments. As shown by Donnelly and Whoriskey (1993), Atlantic salmon displayed limited abilities to acclimate their colouration to match their surroundings. In the larger context of wild Atlantic salmon, the colouration of parr marks within populations are adaptive to the colour of the pebbles and gravel in streams in order to avoid the visual detection by predators. These studies show that a difference in number and contrast of parr marks can make individuals more or less suitable to a particular environment (Donnelly and Whoriskey, 1993). This leads to conservation issues in regard to the reintroduction of salmon into the wild. As salmon are completely absent or present in very small numbers in previously populated rivers, captive bred salmon are beginning to be introduced into the wild to compensate for depleting wild stocks (Parrish et al., 1998; Youngson and Verspoor, 1997). However, as it has been shown that the colouration and contrast of salmon parr marks is influenced by environmental factors, predators quickly prey upon the introduced fish that are not as well suited to their environment as native fish (Kawamura et al., 2012). As parr mark number and colour is at least in part genetically controlled (Kudo et al., 2002), then if the loci controlling a large proportion of the variation in phenotype can be detected and mapped, future conservation efforts can utilize the information in order shift the mean phenotype of a population to be more suitable for a specific environment. 2

11 1.4 A Brief History of Selection in Livestock Species Humans have been using selective breeding techniques for as long as animals have been domesticated. In livestock practices, there are many ways that selection is implemented, many using phenotypic and pedigree information. An example being the Best Linear Unbiased Prediction (BLUP), that is traditionally used to determine an animal s estimated breeding value (EBV) in order to rank the most superior individuals for breeding purposes (Oltenacu and Broom, 2010; Robinson, 1991). Changes across generations using breeding models based on pedigree and phenotypes have been successful throughout history (Donaldson and Olson, 1957; Gjedrem, 2012; Osborne, 1957); however, such techniques have limitations regarding the interplay between genetic and environmental factors associated with quantitative traits. Quantitative traits are those with continuous phenotypic variation that are most commonly controlled by multiples genes and by environmental factors. Due to their complex nature, the observed phenotypes of quantitative traits do not necessarily reflect their underlying genetic component; therefore they are generally very difficult to select using traditional selection methods (Collard et al., 2005). The change across generations using phenotype and pedigree based breeding models are also limited for animals that have long generation times; with generation intervals for important livestock species varying from 1.5 years in pigs, to 4-5 year in dairy cattle, and over 6 years in horses (Gandini et al., 2004). While the Atlantic salmon has a moderate 3-4 year generation interval, results of selection are still limited, as change occurs faster with shorter generation times. While significant advances have been made in production yield of Atlantic salmon since it was first cultured, efforts are limited without considering the genetic basis of quantitative traits. This leads to a new era wherein genetic information is included in breeding models to attempt to overcome the limitations of the use pedigree and phenotypic information alone (Taylor, 2013). 1.5 Introduction to Genetic Markers, QTL Mapping, and Linkage Mapping Modern genetic markers are DNA sequences found in both coding and non-coding regions at regular intervals throughout the genome. In response to the need for the combination of phenotypic and genotypic information in breeding models, the utilization of molecular markers has become common practice for detecting quantitative trait loci (QTL). QTL are regions along the chromosome that control or are closely linked to the genes affecting 3

12 quantitative traits. Up until the 1980 s, the study of quantitative traits was done without the use of genetic information, but rather through the use of statistics alone (see review by Kearsey and Farquhar, 1998). The first genetic markers to be used in the detection of QTL were restriction fragment length polymorphisms (RFLPs) (Beckmann and Soller, 1983), however were limited in the number of markers available (having to rely on restriction endonuclease recognition sites), and were very costly and labour intensive to develop. Other techniques utilizing different types genetic markers have been more successful in mapping QTL, which due to the development of the Polymerase Chain Reaction (PCR) are more efficient and cost effective than RFLPs. Early examples of PCR based molecular markers used for QTL mapping include: random amplified polymorphic DNA (RAPD) as in studies on Eucalyptus species (Rattapaglia and Sederoff, 1994), and cotton (Lin et al., 2005), amplified fragment length polymorphisms (AFLPs) as shown in rats (Otsen et al., 1996) and genetic mapping in larch species (Arcade et al., 2000), as well as microsatellites as used in wheat (Somers et al., 2004) and rainbow trout (Haidle et al., 2008). While the aforementioned molecular markers are successful in mapping QTL in a variety of species, the use of single nucleotide polymorphisms (SNPs) is becoming increasingly more popular. SNPs are single base pair mutations occurring throughout the genome. SNPs may be employed for a variety of purposes including genome-wide association studies (Burton et al., 2007) pharmacogenomics (McCarthy and Hilfiker, 2000), and QTL mapping (Pletcher et al., 2004). The prevalence of SNPs throughout the genome leads to a greater probability that markers will be closely linked to genes of interest, an area of great interest in QTL detection. Due to the development of high-throughput SNP genotyping, SNP analysis is becoming increasingly efficient and cost-effective (Yu et al., 2011). While SNPs are less informative at low densities than other genetic markers such as microsatellites, their abundance and analytical efficiency outweigh this cost when performing high-density genotyping (Jehan and Lakhanpaul, 2006). In Atlantic salmon, the development of high-throughput 6K SNP chips (Kent et al., 2009) has allowed estimation of moderate density linkage maps in both the North American and European subspecies (Brenna-Hansen et al., 2012; Lien et al., 2011) and currently a 200K Atlantic salmon chip is under development. 4

13 Linkage mapping is based on the recombination frequencies between markers, working under the premise that the lower the recombination frequency between two markers, the closer together they are along the chromosome, and vice versa. The distance based on recombination frequencies are outlined in the units of centimorgans (cm). Linkage mapping is different from physical mapping, which outlines the actual physical distance in base pairs of DNA markers along a chromosome. Linkage maps are invaluable tools in the detection of QTL, as the recombination frequency between markers is the means for estimating the location of the markers associated with significant QTL on each chromosome. 1.6 Marker Assisted Selection There are various ways in which the locations of QTL may be implemented for practical industry use; marker assisted selection being one of these. Marker assisted selection (MAS) works under the premise of selecting a genetic marker that is linked, or closely linked to a QTL of interest. In this way the traits of interest are indirectly selected based on genetic markers, rather than the phenotype itself. MAS has been variably successful, with genetic gains seen in plants (see review by Young, 1999) and some success in livestock species (Dekkers, 2004). The applications of MAS hold much promise for the future. MAS has the benefit of not only increasing the accuracy of EBVs, but can allow identification of animals with high EBVs at an earlier age (Dekkers, 2004). The accuracy of EBVs increases with the age of the subject due to increasing numbers of phenotypic records for it as well as its relatives. If the use of molecular markers in the selection process can aid in selecting superior families at an earlier age, this can minimize the costs associated with raising large numbers of candidates with potentially low EBVs. MAS can be used to screen individuals as soon as their genotype information is available in order to sequester those with the highest EBVs. Whether the benefits of MAS for livestock improvement outweigh the development costs is still under debate. Theoretically, MAS can be extremely useful in the selection of quantitative traits and traits with variable phenotypes, as it utilizes genomic information in supplement to phenotypic records (Collard et al., 2005). However, the costs involved in genotyping enough informative marker loci can be very high. There are two very important steps involved in developing markers for MAS; high resolution mapping, and the validation of markers, both of 5

14 which can be very costly to researchers (Collard et al., 2005). Due to the availability of a 6K SNP chip for Atlantic salmon, developing a high density of polymorphic SNP loci can be relatively cost-effective. Marker loci that are evenly distributed along the chromosomes with a maximum distance of 10cM between loci can theoretically allow for the distinction of multiple QTL with small effects and single QTL with large effects. Validating markers for MAS can be the limiting factor in many QTL studies, as even with high densities of genetic markers, the associated loci are not always reliable in determining phenotype (Collard et al., 2005). There is always the chance that markers associated with quantitative traits that are reliable in the population for which they were developed may not be informative in different populations or environments (Reyna and Sneller, 2001). Therefore large amounts of preliminary testing of the reliability of genetic marker in predicting phenotype must be done in order to implement MAS in modern breeding programs. Despite the potential costs associated with MAS, there is high optimism about the use of SNPs for MAS (Rafalski, 2002). The availability of PCR multiplexes, allowing the genotyping of multiple loci in one reaction, has also improved the efficiency and costs associated with highdensity SNP analysis (Donini et al., 1998). As discussed previously, the availability of an abundance of SNP loci, as well as the increasingly affordable costs of high-throughput SNP genotyping makes MAS using SNP loci to be a realistic objective in the future. 1.7 North American and European Atlantic salmon Subspecies Between million years ago, the common ancestor of all salmonid species underwent a whole genome duplication event (Allendorf and Thorgaard, 1984). Salmonids are now in the process of returning to a diploid state. Within salmonid species, there are significant variations in chromosome number. For example, rainbow trout have a total chromosome arm number (NF) of , Arctic charr with NF=96-98 (Phillips and Ráb, 2001), whereas Atlantic salmon vary from 29 chromosomal pairs (NF=74) in the European subspecies, to 27 chromosomal pairs (NF=72) in the North American subspecies. Genetic distinction between the two subspecies has been determined in numerous ways, such as microsatellite differentiation (McConnell et al., 1995) and variation in mitochondrial DNA (Bermingham et al., 1991); however only recently it has been discovered specifically how the karyotypes of the two 6

15 populations differ. In 2012, Brenna-Hansen and colleagues discovered through the use of fluorescence in situ hybridization (FISH) analysis that three chromosomal rearrangement events have occurred in the North American subspecies. It was proposed that chromosomal translocation resulted in the fusing of the p-arm of chromosome 1 (Ssa01) with Ssa23, the fusion of Ssa08 to Ssa29, as well as Ssa26 with Ssa28. During these translocation events, the study proposes that the q-arm of Ssa01 was left behind, resulting in a new acrocentric chromosome and thus explaining the NF of 72 rather than 74 as in the European subspecies. Though these rearrangements are significant, it was determined that a high proportion of SNP loci developed for the European subspecies are still informative in North American strains, indicating DNA conservation (Brenna-Hansen et al., 2012). It is therefore unknown how chromosomes may segregate if the two-sub species are crossed, specifically in regard to the three chromosome pairs in North American Atlantic salmon that underwent significant rearrangement. 1.8 Objective, Hypothesis and Predictions. It is the main objective of the study is to detect QTL associated with parr mark number and contrast, as well as for size (length and weight) and shape (morphometric landmarks) in F 2 transatlantic salmon. The fish used in the present study are transatlantic F 2 s. Although the fish in the 2008 study by Boulding et al. were transatlantic backcrosses, their sires were full-siblings to the parents of the F 2 s. Therefore, I expect some similarity in the location of the detected QTL, except on chromosomes Ssa01/Ssa23, Ssa08/Ssa29 and Ssa26/Ssa28, due to the large difference between the European and North American subspecies, and therefore possible drastic differences in allele segregation (Brenna-Hansen et al., 2012). It has been shown that despite the differences in linkage group number, marker sequences are relatively well conserved between the two subspecies (Brenna-Hansen et al., 2012). Therefore, if marker sequences are well conserved between North American and European Atlantic salmon, then many of the detected QTL will be in similar locations to those found in the transatlantic backcross hybrids of the Boulding et al. (2008) study. However, due to the presence of fused chromosomes in the North American subspecies, as well as novel allele segregation in the F 2 population I expect more differences in 7

16 detected QTL in comparison to either purebred strain, specifically QTL mapped to Ssa01/Ssa23, Ssa08/Ssa29 and Ssa26/Ssa28. Recombination in Atlantic salmon occurs at much higher frequencies in females than in males, where it is generally restricted to the telomeres (Lien et al., 2011), whereas recombination in females occurs all along the middle of chromosomes (Moen et al., 2008). In the F 1 parents, segregation and recombination would therefore be occurring between chromosomes from different subspecies in both males and females, rather than only in the four hybrid sires used in the backcrosses by Boulding et al. (2008). Following Mendel s law of segregation stating that each homologous chromosome has an equal probability of being passed on to the next generation (Snustad and Simmons, 2006), it is expected that the F 2 progeny would display transgressive segregation which can be inferred based on greater ranges of observed phenotypes, specifically size, shape, and colouration. Furthermore, large QTL that are not variable in ancestral purebred populations should begin to segregate in the F 2 population because of segregation creating individual progeny with both homologs for a chromosome from the same purebred grandparent and also because of crossover in the F 1 hybrid females. The St. John aquaculture strain (North American) and the Mowi aquaculture strain (European) of Atlantic salmon are known to differ in growth rate, size at maturity, shape, parr mark number and coloration and spotting (B. Glebe, pers. comm., Boulding et al., 2008) that are likely because of genetic differences at the QTL determining these traits among their wild founder population(s). Therefore, if transgressive segregation is occurring in the F 2 population, then a greater number of significant QTL will be detected in my study relative to previous studies on both purebred Atlantic salmon and backcross transatlantic salmon. 8

17 2.0 Materials and Methods 2.1 Creation of F 2 trans-atlantic hybrid crosses European Mowi granddam and Canadian Saint John River grandsire were crossed in 2001 to create an F 1 full-sib trans-atlantic hybrid family. Fish were spawned using the dry method, with eggs being stripped from the female, and sperm being manually added and the gametes gently mixed. In January 2011, eight trans-atlantic full-sibs (four male, four female) were spawned in the same manner as the grandparents to create four F 2 trans-atlantic hybrid families. The F 2 offspring hatched in March 2011, with three of the four F 2 families surviving. 2.2 General Husbandry All fish were kept and maintained at the Saint Andrews Biological Stations (SABS) (Department of Fisheries and Oceans Canada) in Saint Andrews, New Brunswick, Canada. The F 2 eggs were under constant shading and held at 5-6 o C for days. Any dead eggs were removed daily based on discolouration, and deformities using a glass pipette and suction bulb slightly larger in diameter than the eggs. After approximately 60 days, when almost ready to hatch, all eggs were transferred to shallow hatching tanks (5 feet in diameter, 8 in depth, 100L volume). Hatching occurred over 14 days and alevins were kept at 6 o C for 1 month until yolk sac was depleted. As alevins and naturally seek coverage (Thorstad et al., 2011), matts were added to the tank in order to provide shading for the juvenile fish. Fish were kept in six tanks, two for each family with approximately even fish densities. Once the yolk sac was almost completely absorbed, the alevin/fry began to swim to the surface, indicating the beginning of feeding. At this time, the temperature of the water was raised 1 o C per day to a maximum of 10 o C in order to stimulate feeding. Fry were mostly fed live Artemia nauplii at the beginning of feeding, with small amounts of dry feed ( μm). Once feeding was consistently observed, fry were weaned of Artemia and switched to primarily dry feed given daily. Fry were maintained under regular photoperiod at 10 o C until the ambient 9

18 temperatures during summer naturally raised that of the water (approximately o C). Wastes were removed via circular flow to a central drain, which was flushed daily. In the fall of 2011, when average fish weight reached 3-5grams, all fry were transferred to 1000L tanks using a soft mesh net. As SABS was under construction during the duration of this study the fish were kept outside under protective tents. At this time families were combined, and all fish were kept in three 1000L tanks, one for each family. Water was maintained at ambient temperature, which due to the outside temperatures in the winter resulted in variably lower water temperatures than is common practice in aquaculture (S. Leadbeater, pers. comm.). Survival was not affected, however growth was observed at a slower rate than fish kept under ideal conditions. Feeding was variable based on the habits of the fish (target was of 3.5-5% body weight per day). Technicians monitored feeding in order to allow fish to feed to satiation without allowing unnecessarily large amounts of uneaten food and wastes to accumulate that might cause issues in hygiene. The size of the pellet feed increased dependent on the size of the fish. In March 2012, all remaining fish were transferred to two indoor 9000L tanks and maintained at ambient freshwater conditions. As at this time fish had already been PIT-tagged, individuals from all families were randomly assigned to tanks. When fish were an average of 50grams, water was converted to 10ppt salt to avoid fin erosion and reduce freshwater use; 10ppt has not been shown to affect the incidence of smoltification (S. Leadbeater, pers. comm.). At the time of the final measurement fish were being maintained at ambient temperature (approximately 2 o C in winter) at 10ppt. 2.3 SNP Development Two separate SNP multiplexes were used in this study. The first multiplex had been used to genotype the transatlantic backcross families B-D (Boulding et al., 2008). This multiplex was designed using Spectro-DESIGNER v3.0 from Sequenom and consisted of 129 SNPs chosen from a larger subset that had been discovered by aligning EST contigs (Hayes et al., 2007). It was assumed that since the parents of the backcrosses were full-sibs with the parents of the F 2 s, a high proportion of SNPs polymorphic in the backcross families would also be informative in the F 2 crosses. 10

19 Extracted DNA from the grandsire and six parents were sent to the Centre for Integrative Genetics (CIGENE) in Aas, Norway for genotyping with Illumina 6K Atlantic salmon SNP chip. Each SNP was individually analysed and SNPs that displayed proper segregation and high degree of polymorphism were noted. At the CIGENE facilities 329 preferred SNPs were chosen. SNPs were chosen at a minimum distance of 10cM from each other. SNPs were chosen based on loci with the most variability in the parent genotypes, as well as avoiding multisite variants (MSVs) when possible. MSVs in Atlantic salmon are loci that are tetraploid rather than diploid (Gidskenhaug et al., 2011), and therefore complicate SNP analyses with a larger number of genotype combinations. Larger densities of SNP loci were deliberately placed at the centre and near the telomeres of chromosomes as well as on chromosomes that are fused in the North American subspecies. Primers were designed using Spectro-DESIGNER. Of these chosen 329 SNPs, 121 that gave good coverage of the genome and could be amplified in one of four PCR multiplexes were selected for genotyping in the F 2 population. 2.4 DNA Analysis DNA was extracted from fin clips using Qiagen DNeasy blood and tissue kit according to the manufacturer s specifications. DNA quality was checked by running samples in separate lanes on a 1% 150mL agarose gel next to lanes containing high DNA mass ladder and λ DNA at concentrations of 3.75, 25, 30 and 60 ng/ml as standards to approximate sample concentration. Gels were run for 45 minutes at 180V. These quality checks were performed on randomly selected samples from each 96 well plate. DNA of samples were all quantified using Nanodrop technology. Samples for the Sequenom assay were then standardized to approximately 10ng/mL. Samples from family 520 (a wild European and North American backcross) were used as preliminary samples for testing of the new multiplex. The F 2 progeny were too young at the time of multiplex design to PIT-tag, therefore any DNA sampling at this age could not be connected to phenotypic data obtained at a later age. Family 520 was therefore used as it most closely simulated the genotypic variation that may be displayed in a subspecies cross. This was done in 11

20 order to generally assess whether the SNP loci in the multiplexes would be polymorphic, and therefore informative, in a transatlantic cross. Primers were brought to the Clinical Genomics Centre at Mount Sinai Hospital in Toronto, Canada. All F 2 samples were genotyped using Sequenom MassARRAY iplex Gold Assay with four different multiplexes of each sample. The Sequenom MassARRAY iplex Gold Assay works as a primer extension process specifically designed to detect single nucleotide differences in sequence. It is the difference in mass between specific alleles of extension products that allows the software to assign different SNP alleles. The end product provides accurate and scorable SNP genotyping. Full details can be found in Gabriel et al. (2009). SNPs were analysed and personally verified on Sequenom Spectro-TYPER for any miscalls which may have occurred. SNP loci were deemed failed and removed from further analysis if the majority of samples did not amplify or if the loci significantly deviated from expected Mendelian segregation patterns making it uninformative. 2.5 Morphometric Data During November 15 th -25 th, 2011 once fish of the three F 2 families were an average weight of 8-9grams, all fish from the population were non-lethally anesthetised (using tricaine methanesulfonate (TMS) at 0.07g/L). Anesthetised fish were then photographed for length and morphometric data, weighed (wet weight to the nearest 0.1g), and fin clipped at SABS. Fin clips were kept in 1.5 ml tubes with o-ring screw caps, filled with 0.5mL of 95% EtOH at 4 o C. Phenotypic data was obtained from 1047 fish during the first measurement period; at this time all but 600 fish were euthanized with a lethal dose of TMS (0.7g/L). In order to keep the families as large as possible, approximately 300 individuals were kept from crosses 1 and 3; all of family 4 were euthanized. During November 28 th -30 th, 2012, when fish were an average of 100g and beginning to display signs of undergoing the parr-smolt transformation, they were measured for a second time. Of the 600 fish that were retained, 499 survived to the second measurement period. Fish were anesthetised in the same manner as the first measurement, and were once again weighed (wet weight to the nearest 0.5g) and photographed for morphometric analysis. 12

21 Photographs of individual fish were digitized using tpsdig (Rohlf, 2010) and 12 landmarks were recorded (Table 1, Figure 2). Each photograph was individually scaled, accounting for potential variation in zoom of the photos. All photographs were scaled, aligned and rotated using tpsregr 1.37 (Rohlf, 2009), thereby being standardized as centroid size, maintaining the geometry of the landmark positions. Centroid size was used as a covariate in order to determine variation in landmark position based on shape, rather than size. Both x and y coordinates of each landmark were analysed as to specify the directional variation (vertical vs. horizontal) for the specific anatomical feature. An example of variation in landmark position can be visualized from Figure 3. This method of geometric morphometrics analysis allows for a statistical test of the association between individual phenotypes of each landmark and the genotypes of the individual; a crucial component in the detecting of QTL associated with complex traits. After all photographs had been digitized, errors were determined using aligned coordinates in tpsregr 1.37 (Rohlf, 2009) removing any outliers, and re-digitizing any mistakes in landmark position. For both measurement periods, landmark AnPCF, the most anterior point of the pectoral fin, was removed due to the extremely high degree of variation in landmark positioning. As the fish were anesthetised at low concentrations in order to avoid the incidence of over dosage, the pectoral fins were often mobile. At the second measurement period, due to living in tank conditions, fin erosion was prominent throughout the population. Therefore the unreliability of the landmark would not give accurate QTL results, and was removed from further analysis. The contrast between skin and parr marks was quantified from the parr photographs used for morphometric digitizing (Figure 4). Using Adobe Photoshop 7.0, the eyedropper tool was used to measure the brightness (L/A/B) of a 5x5 pixel area. The brightness was measured from the centre of the parr mark at the lateral line (or the symmetrical centre of the parr mark if not along the lateral line) and on the skin in between parr marks along the lateral line. Individual values were recorded for the brightness of each parr mark as well as the adjacent skin. Relative contrast was calculated as a ratio of each parr mark to skin brightness, and then averaged for each individual fish. All statistical analyses on raw data were performed using StatPlus:mac in Microsoft Excel or R, and were interpreted using graphs created in R. 13

22 2.6 Linkage Map A novel linkage map was created for the F 2 population, as recombination frequencies were found to differ significantly from both the pure North American (Brenna-Hansen et al., 2012) and European populations (Lien et al., 2011). Separate male and female maps were created using JoinMap 4.0 ( Marker grouping was done at a minimum LOD of 3.0. Linkage maps were first created separately for each family and sex, and then combined into two sex-specific maps incorporating all crosses. The final female map was 972 cm in length, while the male s was cm. 2.7 QTL Detection GridQTL software was used in order to detect QTL for the trait of interest ( GridQTL is an online web application used for mapping QTL utilizing phenotypic and genotypic data (Hernández-Sánchez et al., 2009). The BC-F2 portlet was used for all analyses in steps of 1.0cM. This module provides additive, and dominance genetic effects, and is desirable as it can be used with marker information on a select few chromosomes as opposed to genome wide. GridQTL utilizes algorithms to perform regression analyses in order to detect QTL as in its earlier version, QTL Express (Seaton et al., 2006). Experiment-wide permutations with 250 iterations were performed, at α=0.05 and α=0.01 for all analyses. Five separate QTL analyses were performed as follows: size data (length and weight) at both measurement periods; landmark data at both measurement periods; and parr mark data. Given that both male and female-specific linkage maps were used in the analysis, a total of 10 different QTL analyses were undertaken. PEV, the percentage of the phenotypic variation explained by a particular QTL, was estimated as: PEV = 100 (1 F_RMS / R_RMS) where F_RMS is the residual mean square for the full model and R_RMS is the residual mean square for the reduced model in GridQTL (Haley et al., 2009; S. Knott pers. comm. to EGB). 14

23 3.0 Results 3.1 Phenotype statistics Length and weight were weakly correlated in young parr (r=0.41, P<0.001, N=1047) with length only explaining 16% of the variance of weight. Consequently it was therefore decided to analyse length and weight as separate traits. Length and weight were more highly correlated in fish near smolting (r=0.90, P<0.001, N=499); nevertheless they were also analyzed as separate traits for consistency. During the first measurement period, lengths across all families ranged between cm (mean±se, 9.05cm ±0.04), and weights varied between grams (mean±se, 8.74g±0.12) (Table 2). Both traits approached a normal distribution across all three families (Figure 5, Figure 6). Significant differences were found among the three crosses for both traits (one-way ANOVA, P<0.001 df=2), with individuals of cross 1 being significantly shorter and lighter than the other two families (Tukey post-hoc, P<0.001). The two families maintained through the second measurement period, had total lengths ranging from cm (mean±se, 19.18±0.14), and weight ranging from grams (mean±se, 95.10±1.73) (Table 2). Both traits displayed relatively normal distributions across all three families (Figure 7, Figure 8). Weight was significantly lower (Welsh s t-test, P<0.001 df=495) and length was significantly shorter (Welsh s t-test, P<0.001, df=495) in cross 1 than in cross 3. The number of parr marks per individual at the first measurement period ranged from 6 to 12, (mean±se, 9±0.03, Table 2) and were normally distributed (Figure 9). The relative contrast of skin colouration to parr mark pigmentation ranged from (mean±se, 1.88±0.01), (Table 2). The distribution of skin/parr mark colouration was positively skewed, with few individuals showing high contrast, and a greater number with lower contrast values (Figure 10). The data was not transformed in order to retain the natural outliers seen in regard to colour variation. Significant differences between families for both parr mark number and contrast was determined (one-way ANOVA, P<0.001, df=2). It was found that cross 3 had significantly more parr marks 15

24 than the other two families, and also a significantly higher contrast between skin and parr mark colouration (Tukey post-hoc, P<0.001). At the second measurement period, it was observed that there was a large variation in the life stages as well as colouration displayed by the F 2 s. Fish near smolting, characterized by a streamlined body shape, and a lack of parr marks, displayed a range of silver-type colouration, some with darker blue or green hues, and others with being very vivid silver. Smolts also display black speckling at their dorsal end, which was also very variable within the population. This type of colour variation was also seen in fish still at the parr stage, with very distinct colour differences (Figure 11). 3.2 QTL detection, length Length QTL Female map Several QTL were detected that were highly significant at α=0.01 at the experiment-wide level, and accounted for large proportions of the phenotypic variation (PEV) associated with length in the female F 2 s (Table 3). The largest QTL for length at an early age were detected on Ssa01/Ssa23, Ssa21, and Ssa26/Ssa28 explaining 6.4%, 5.8%, and 5.4% of the phenotypic variance, respectively. Ssa02, Ssa09 and Ssa25 also contained highly significant QTL accounting for 4.2%, 2.3%, and 2.4% PEV, respectively. Two smaller QTL were mapped to Ssa03 and Ssa07 explaining 1.7%, and 1.8% PEV, respectively (Table 3). Highly significant QTL detected for length at later stages were mapped to Ssa01/Ssa23 (6.4%), Ssa02 (5.3%), Ssa03 (5.4%), Ssa11 (6.4%), and Ssa21 (6.2%), and Ssa26/Ssa28 (4.3%), with one smaller significant QTL (α=0.05 at the experiment-wide level) being detected on Ssa09 (3.2%) (Table 5). Some similarities arose between the QTL detected for early and late length, most notably QTL on Ssa26/Ssa28 at both stages being detected at similar positions of 8cM at measurement period one, and 6cM at measurement period 2. Similarities were also noted between QTL at both stages on Ssa02, Ssa03, Ssa07, Ssa09, and Ssa21, though the positioning of the QTL varied more widely than Ssa01/Ssa23. All length QTL mapped at the later stage were also detected in young parr, however QTL for early length on Ssa25 was not detected in older individuals. 16

25 3.2.2 Length QTL Male map The use of the map based on male meioses detected three highly significant QTL (at the experiment-wide level at α=0.01) for early length in the three F 2 crosses, accounting for large proportions of the phenotypic variation (Table 4). QTL were mapped to Ssa01/Ssa23 (6.2%), Ssa07 (2.5%), and Ssa09 (4.4%). An increase in the number of QTL detected was seen when analysing length at the second measurement period (Table 6). The largest QTL all accounting for more than 5.5% of the phenotypic variation were mapped to Ssa01/Ssa23 (6.0%), Ssa09 (6.2%), Ssa16 (5.6%), and Ssa21 (6.5%). Five smaller QTL detected at the experiment-wide level at α=0.05 were found on Ssa06, Ssa07, Ssa11, Ssa12, and Ssa24, accounting for % of the phenotypic variance. All QTL for length at an earlier age were also detected in older fish, many in similar positions. Length QTL in younger fish on Ssa01/Ssa23 was found at the same position of 32cM in the older age group. Several QTL were mapped to very similar positions in both age groups such as on Ssa07, where length QTL were detected at 44cM in young fish and 43cM when older, as well as on Ssa09 where QTL for early length was detected at 2cM, and QTL for late length at 0cM. Similarly, QTL on Ssa21 were detected at 1cM at first measurement, and 4cM at the final measurement. Unique QTL on Ssa06, Ssa11, Ssa12, Ssa16 and Ssa24 were found in fish near smolting, but were not found in young parr Comparing length QTL detected with female and male maps QTL for length at an early age were detected in eight locations with the female map, and four with the male map. QTL for early length on Ssa02, Ssa03, Ssa25 and Ssa26/Ssa28 were only detected using the female map, whereas all QTL located with the male map were also found with the female-based map. QTL associated with length at the second measurement period were detected in several unique locations in both sexes, with QTL located on Ssa02, and Ssa03 being exclusive to the female map, and Ssa06, Ssa07, Ssa12, Ssa16 and Ssa24 being detected with the male map only. A total of seven QTL for length in older fish were detected with the female map, and nine using the male map. 3.3 QTL detection, weight Weight QTL Female map Many highly significant QTL for weight were mapped using the linkage map based on female meioses (Table 3). Five of the largest QTL of very high significance for weight at an 17

26 early age were mapped to Ssa01/Ssa23 (6.0% PEV), Ssa02 (5.2%), Ssa11 (4.7%), Ssa21 (5.6%), and Ssa26/Ssa28 (5.0%), with two QTL of high significance that account for lower amounts of the phenotypic variation located on Ssa09 (4.2%), and Ssa10 (3.1%). Three smaller, though still significant QTL (at α=0.05) were detected on Ssa03 (1.8%), Ssa17 (2.0%), and Ssa25 (2.0%). Highly significant QTL for weight in older fish were detected on Ssa01/Ssa23 (8.2%), Ssa02 (5.6%), Ssa03 (8.2%), Ssa09 (5.1%), Ssa11 (5.8%), Ssa21 (8.8%) and Ssa26/Ssa28 (6.9%), (Table 5). One smaller QTL, still significant at the experiment-wide level was mapped to Ssa04, explaining 4.0% of the phenotypic variation. QTL for weight at both measurement periods were detected in similar locations on Ssa09, both ages having QTL positioned at 9cM as well as on Ssa11, with QTL for both early and late weight being found at the 15cM position. QTL on Ssa01/Ssa23, Ssa02, Ssa03, Ssa21 and Ssa26/Ssa28 were detected on the same chromosome at both measurement periods, however there was variation in position. Unique QTL were found on Ssa17 and Ssa25 in young parr, but not in near smolts Weight QTL Male map Two very large and highly significant (α=0.01, experiment-wide) QTL for early weight were detected on Ssa01/Ssa23, and Ssa09, explaining 5.8%, and 5.0% PEV, respectively (Table 4). Six other highly significant QTL detected at α=0.01 at the experiment-wide level were mapped to Ssa02 (1.8%), Ssa07 (4.1%), Ssa10 (2.2%), Ssa11 (4.4%), Ssa21 (2.7%) and Ssa26/Ssa28 (2.0%). One smaller QTL was found at the experiment-wide level (α=0.05) on Ssa12, explaining 1.5% of the phenotypic variation (Table 4). Three very large and highly significant QTL were detected at the second measurement period (α=0.01, experiment-wide) located on Ssa01/Ssa23, Ssa16, and Ssa21 explaining 7.8%, 8.5%, and 9.2% PEV, respectively (Table 6). Four other highly significant QTL significant were mapped to Ssa07 (6.8%), Ssa09 (8.0%), Ssa12 (4.8%), and Ssa24 (5.3%) using the male-based map. Four additional QTL significant experiment-wide at α=0.05 were mapped to Ssa02, Ssa04, Ssa06, and Ssa26/Ssa28, explaining between % of the phenotypic variance (Table 6). Many of the weight QTL detected at the first measurement period, were detected in similar locations at a later age. QTL on Ssa01/Ssa23, Ssa02, and Ssa21 were mapped to the same positions at both measurement periods while QTL on Ssa26/Ssa28 differed by 1cM between young and older fish. Weight QTL were also found in very similar locations on Ssa09 (0cM in young fish and 6cM in older fish) and Ssa12 (0cM at first measurement and 2cM at the second measurement). QTL for early weight on 18

27 Ssa10 and Ssa11 were not detected in older fish, whereas QTL detected on Ssa04, Ssa06, and Ssa24 were not found in young parr Comparing weight QTL detected with female and male maps QTL associated with weight at the first measurement were detected on 10 linkage groups with the female map, and nine with the male map. QTL on Ssa03, Ssa17 and Ssa25 in females were detected in males, whereas QTL on Ssa07 and Ssa12 were detected with the male map only. QTL for weight at the second measurement period on Ssa03 and Ssa11 were only detected with the female map, and QTL on Ssa06, Ssa07, Ssa12, Ssa16, and Ssa24 were only detected when the male map was used, similar to QTL for length in older fish. A total of eight QTL for weight at the second measurement period were mapped with the map based on female meioses, and 11 with the male specific map. 3.4 QTL detection, parr mark number and contrast Parr mark QTL Female map Numerous highly significant QTL associated with parr mark number were detected using the female linkage map (Table 7). Very large and highly significant QTL were mapped to Ssa01/Ssa23 (8.1%), Ssa03 (10.2%), Ssa04 (8.6%), Ssa16 (10.7%), and Ssa21 (9.4%). Other large and significant QTL for parr mark number were found on Ssa02, Ssa11, Ssa12, Ssa13, Ssa14, Ssa24, Ssa25, and Ssa26/Ssa28 accounting between % of the phenotypic variation. Two smaller QTL (significant at α=0.05), were mapped to Ssa09, and Ssa10 explaining 1.5% and 1.9% PEV, respectively (Table 7). Large and highly significant QTL for the contrast of skin and parr marks were found on Ssa03, Ssa04, Ssa16, and Ssa21, accounting for 5.6%, 6.5%, 6.2% and 6.4% of the phenotypic variation, respectively (Table 7). Other highly significant QTL for parr mark contrast were also mapped to Ssa01/Ssa23 (4.4%), Ssa13 (2.3%), Ssa24 (2.9%), and Ssa25 (2.9%), with female meioses. Four additional significant QTL accounting for less than 2.0% of the phenotypic variation were found on Ssa02, Ssa10, Ssa11, and Ssa12 (Table 7). 19

28 3.4.2 Parr mark QTL Male map An unexpectedly large number of significant QTL were detected for parr mark number using the male specific map (Table 8). Five very large and highly significant QTL were mapped to Ssa01/Ssa23 (4.1%), Ssa04 (8.4%), Ssa13 (7.2%), Ssa16 (10.7%), and Ssa21 (5.4%). Six other highly significant QTL for parr mark number were detected on Ssa03, Ssa07, Ssa12, Ssa24, Ssa25, and Ssa26/Ssa28 accounting between % of the phenotypic variance across all three F 2 crosses. Four additional smaller QTL were detected at α=0.05 (experiment-wide) on Ssa09, Ssa10, Ssa11, and Ssa14, explaining % PEV (Table 8). Several very highly significant QTL for the contrast of skin colouration to parr mark pigmentation were detected on Ssa01/Ssa23, Ssa04, Ssa12, Ssa13, Ssa16, Ssa21, Ssa24, Ssa25, Ssa26/Ssa28, accounting for between % of the phenotypic variance (Table 8); the largest, both accounting for 6.2% PEV, were mapped to Ssa04 and Ssa16. Three smaller QTL significant at α=0.05 at the experiment-wide level, were found on Ssa05, Ssa07, and Ssa10 explaining between % of the phenotypic variation (Table 8) Comparing parr mark QTL detected with female and male maps Sixteen QTL associated with parr mark number were mapped using the female linkage map, and 15 with the male map. QTL on Ssa02 was detected with the female map, but not with for the male map. On the other hand all QTL found using the male-based map were also detected with that from females. QTL associated with skin and parr mark colour contrast on Ssa02 and Ssa03 were unique to the female map analysis, whereas QTL on Ssa05, Ssa07 and Ssa26/Ssa28 were unique to the male analysis. For skin and parr mark contrast a total of 13 QTL were detected with the female map whereas 12 were detected using the male linkage map. 3.5 QTL detection, morphometric landmarks Morphometric QTL Female map Numerous QTL for 11 morphometric landmark positions were located at the first measurement period with the female-based map (Table 9). Ssa01/Ssa23, Ssa02, and Ssa21 contained highly significant QTL for at least nine of the 11 landmark positions. Most notably, seven QTL accounted for extremely large proportions of the phenotypic variation; Ssa01/Ssa23 with QTL for dpcfx, explaining 11.9% PEV, QTL on Ssa02 associated with DFx and DFy 20

29 (10.8% and 15.0%, respectively), Ssa16 with a large QTL for landmark DFy (9.9%), Ssa21 also associated with DFy (11.0%) as well as dpcfx (13.3%), and Ssa26/Ssa28 mapping a large QTL for landmark dpcfx explaining 10.6 % of the phenotypic variation. QTL analysis at the second measurement period revealed Ssa01/Ssa23, Ssa03, Ssa21 and Ssa26/Ssa28 to contain highly significant QTL for at least nine of the 11 landmark positions (Table 11). QTL accounting for some of the largest proportion of the phenotypic variation were all associated with landmark vpcfy, located on Ssa01/Ssa23 (18.4%), Ssa03 (21.2%), Ssa04 (14.6%), Ssa21 (24.1%), and Ssa26/Ssa28 (17.4%). A very large and significant QTL for landmark vpcfx was also located on Ssa21, accounting for 16.8% PEV. Five additional QTL explaining between % of the phenotypic variation were detected on Ssa01/Ssa23 for landmarks vpcfx and PUx, Ssa03 for vpcfx (14.4%), Ssa21 for PUx, and Ssa26/Ssa28 for landmarks vpcfx and PUx. Interestingly, many of the most significant landmark QTL were unique to one measurement period. QTL for landmarks vpcfy, and PUx on Ssa01/Ssa23 in measurement period two were not detected at high significance in young fish, though significant QTL for landmark dpcfx were detected at both measurement periods. QTL for landmark vpcfx on Ssa01/Ssa23 that was highly significant in older fish was only significant at α=0.05 in young parr, accounting for a small proportion of the phenotypic variation (1.6%, raw data not provided). While Ssa02 contained QTL for 10 of the 11 landmarks in young parr, only two were mapped in older fish, neither of which were of the highest significance at the first measurement period. Ssa03 and Ssa04 in young parr contained many significant landmark QTL, however did not account for the vertical variation of the posterior ventral insertion of the pectoral fin (vpcfy) which displayed extremely high significance in older fish. Ssa16 in young parr contained many significant QTL most notably for landmark DFy, however no QTL were mapped to this chromosome at a later age. Ssa21 at both ages contained QTL for the majority of the 11 landmarks, both displaying significance for landmarks DFy and dpcfx, however the large QTL for vpcfy and PUx in older fish were not found in young parr. Also, only in younger fish did Ssa21 account for QTL for either the x or y coordinates of all 11 landmark positions. QTL for dpcfy and CPx were found to be very significant at both measurement periods on Ssa26/Ssa28, however significant QTL for vpcfx, vpcfy and PUx found at the second measurement period were not seen in the first. 21

30 3.5.2 Morphometric QTL Male map A large number of QTL across 15 linkage groups were detected at the first measurement in young parr for the 11 landmark positions using the map based on male meioses (Table 10). Ssa01/Ssa23 and Ssa02 contained significant QTL for most landmark positions (10 and 9 of the 11 landmarks respectively). Other linkage groups contained fewer morphometric QTL. QTL accounting for the largest proportions of the phenotypic variation were located on Ssa01/Ssa23 for landmarks dpcfx (12.6%), Ssa02 for DFy (8.1%), Ssa16 for landmarks DFx, DFy, and CPy (9.4%, 9.9%, and 7.4%, respectively), and Ssa21 for landmark dpcfx (7.2%) (Table 10). Highly significant QTL for morphometric landmarks were mapped in 12 linkage groups at the second measurement period (Table 12). The highest number of significant QTL for the x or y coordinates of individual landmarks was seen on Ssa01/Ssa23 (nine of 11 positions), Ssa07 (nine of 11 positions), and Ssa21 (10 of 11 positions). QTL accounting for the largest proportion of the phenotypic variation were mapped to Ssa01/Ssa23 and Ssa07 for landmark vpcfy (16.5% and 16.5% PEV, respectively), and Ssa21 for landmarks vpcfx, vpcfy, and PUx (16.8%, 24.1%, and 14.1%, respectively). Six additional QTL explaining % of the phenotypic variance were located on Ssa01/Ssa23 comprising QTL for landmarks vpcfx and PUx, Ssa04 for vpcfy, Ssa07 for PUx, Ssa21 for landmarks SNx and DFy, and Ssa24 for vpcfy (Table 12). Similar to analyses utilizing the female-based maps, morphometric QTL displaying the highest significance most often differed between the two age groups. The large QTL for landmark vpcfy on Ssa01/Ssa23 was only found at the first measurement period, while QTL of very high significance for landmark PUx was unique to older fish. In contrast, a large number of the total landmark positions mapped to Ssa01/Ssa23 at both measurement periods (10 of 11 at measurement one, nine of 11 at measurement 2); as well QTL for landmarks vpcfx and dpcfx were mapped in high significance at both measurement periods to this linkage group. Nine of the 11 landmark positions had significant QTL on Ssa02 in young parr, however no QTL of significance were mapped in older fish. The large QTL for landmark vpcfy on Ssa04 was found to be unique to older fish, as well as QTL for vpcfy and PUx on Ssa07. Many significant QTL were mapped to Ssa16 in young fish with three accounting among the largest PEV (DFx, DFy, and CPy), however no QTL of significance were mapped to this linkage group in older fish. 22

31 Ssa21 contained many significant QTL at the two measurement periods, both showing significance for landmark DFy, CPx, FKx and y, vpcfx, dpcfx, and PUx Comparing morphometric QTL detected with female and male maps Many of the largest QTL for morphometric landmark positions at the first measurement period were similar between male and female maps (Table 9 & 10). While many of the most significant QTL detected with the male map were also found using the female map, the highly significant QTL for dpcfx on Ssa26/Ssa28 was found with the female map only. The largest difference between the female and male-based results for measurement two was the number of significant QTL detected on Ssa03, where eight landmark positions were mapped with the female map, and only one with the male map. Similar results for landmark position were seen at the second measurement period, where the majority of the largest QTL were consistent between male and female maps (Tables 11 & 12). Some exceptions arise such as with significant QTL for landmarks vpcfx, and PUx on Ssa26/Ssa28 being unique to the female-based map, and landmark PUx on Ssa07 only being unique to the male map. Notable differences also occurred between male and female maps in respect to the number of landmark QTL being seen on Ssa03, Ssa07, and Ssa26/Ssa28. QTL for 9 of the 11 landmarks were detected on Ssa03 when the female map was used, however only two were detected when the male map was used. Ssa07 contained QTL for 9 different landmark positions (x or y coordinates) in the male-based map, but only two with the female map. Finally, 10 of the 11 landmark positions had significant QTL mapped to Ssa26/Ssa28 with the female map, however only one had significant QTL when the male map was used. 23

32 4.0 Discussion To the best of my knowledge, this is the first ever creation of an F 2 transatlantic cross between North American and European subspecies of Atlantic salmon in order to detect QTL. The success of this novel cross has led to many interesting, albeit unexpected, results. 4.1 Colour Related Traits Qualitative look at colour variation It was predicted that the creation of an F 2 transatlantic cross would display transgressive segregation because of recombinant events between the chromosomes from the two subspecies. Based on the results of both genetic and phenotypic records this can be said to hold true. Looking at the colour types displayed at the second measurement period, the variation in markings and shades is quite astounding. Not only were there dramatic differences in colour and pattern variation between fish still at the parr stage and those having visibly smolted, but also within these two categories differences in colouration were prominent. More interestingly however was the presence of red spots of the lateral sides of some parr that varied in intensities. It is uncommon for North American aquaculture strains to display this colouration (B. Glebe, pers. comm.), however it is common in wild Atlantic salmon. Blanc et al. (1994) showed that red spots along the lateral sides of brown trout (Salmo trutta) have an approximate heritability of 0.7, indicating strong genetic control. As both Atlantic salmon and brown trout are of the family Salmonidae, and currently classified within the genus Salmo, the red spots may be under a similar type of genetic control; therefore the presence of red spots in Atlantic salmon parr may have similar inheritance, though this has yet to be investigated. Future study on the quantification of colour variation and its relationship between families using the F 2 photographs could lead to very interesting results An examination of the genetic components of colour variation When examining data for parr mark number and colouration, my results indicate that both traits are controlled by multiple loci. Highly significant QTL for parr mark number were detected on multiple linkage groups. My hypothesis and predictions relating to the locations of detected QTL were based on the findings in Boulding et al. (2008) of transatlantic backcrosses. I 24

33 expected results to vary slightly between the two studies, specifically I expected QTL mapped to the chromosomes fused in the North American subspecies to differ from the backcross population, I also expected to detect a greater number of significant QTL loci in the F 2 s in comparison to the backcrosses. This hypothesis can be upheld based on the large number of detected QTL in the F 2 generation, though the location of some QTL were not consistent with that of Boulding et al. (2008). While the study by Boulding mapped three novel locations for parr mark number using a male-based map, I detected 15. Furthermore only one of the three chromosomes found to be associated with parr mark number in the backcross population was found to be the same in the F 2 s. In the F 2 population, no significant QTL were detected on Ssa08 and Ssa17 as in the backcrosses, however a large QTL was found on Ssa01/Ssa23 in compliment to the QTL mapped to Ssa23 in Boulding et al. (2008). As it is unknown how Ssa01/Ssa23 are segregating in the F 2 s, especially due to the presence of the lone q-arm of Ssa01 in North American Atlantic salmon, when comparing to the backcross population, I will not be making distinctions between the fused chromosomes 1 and 23, 8 and 29, or 26 and 28. For example, the QTL detected on Ssa23 is considered complementary to QTL detected on Ssa01/Ssa23 in the F 2 s given the fact that the fusion between Ssa01 and Ssa23 was previously unknown. Similar to the results of the parr mark data, a large number of QTL were detected for skin/mark contrast. Once again comparing to the results of Boulding et al., (2008), it was hypothesized that the F 2 data would result in QTL in similar locations to those of the backcross populations, with additional novel QTL when using the male-based map. As expected, a large QTL associated with contrast values was mapped to Ssa07 in both studies, however the QTL located on Ssa02 in the backcross families was not seen using the male-based F 2 map. QTL associated with contrast was however detected in the F 2 s on Ssa02 with the female map. While these results are relatively complementary between the two studies, a total of 13 and 12 QTL for contrast were detected with both the male and female F 2 maps, respectively. As seen with the parr mark data, this is a much larger number of detected QTL, and also explains a larger proportion of the phenotypic variation than was previously found. Previous studies regarding pigmentation in fish have frequently involved supplementing the study species diet with carotenoids. For example, Gouveia and Rema (2005) found that 25

34 augmented concentrations of carotenoids alter the skin pigmentation in ornamental goldfish (Carassius auratus), similar to the results of Kalinowski et al., (2005) where increasing levels of astaxanthin was correlated with an increase in skin pigmentation of the red porgy (Pragus pragus). Contrary to these studies, Storebakken et al., (1987) found that carotenoid equivalents (astaxanthin, astaxanthin dipalmitate and canthaxanthin) had an effect on the flesh colour of Atlantic salmon, however not skin pigmentation. These results, along with the equality of feed amounts and types for the F 2 s in all tanks does not lead me to believe that nutritional content is the reason for the variation of colour phenotypes seen. Similarly, while parr mark contrast does have an environmental component (Donnelly and Whoriskey, 1993), due to the estimation of tank effects in my experimental design, I do not believe that environmental conditions are significantly affecting parr colouration in the F 2 s. The large number of QTL associated with parr mark number and contrast is most parsimoniously explained by the polygenic control of colour-related traits. It was already shown that parr mark number is partially genetically controlled (Kudo et al., 2002), and that salmon have limited abilities to acclimate the intensity of their parr marking to their environment (Donnelly and Whoriskey, 1993). In this respect, the number of QTL for both traits suggests they are polygenic. This explanation is supported through a study by Greenwood et al. (2011) conducted on threespine sticklebacks (Gasterosteus aculeatus), which concluded that based on QTL analysis, the control of pigmentation is complex in nature. However, Kawamura et al. (2012) noted that bottlenecking and low genetic diversity was the cause of the fixation of QTL associated with spotting in the amago salmon (Oncorhynchus masou ishikawae). Therefore due to the high selective pressures put upon both the North American and European subspecies of Atlantic salmon for aquaculture, loci affecting colour traits may have been fixed. The F 2 population therefore may have returned segregation at the previously undetectable loci. I believe it is a combination of the two, that both traits are controlled by multiple loci, and due to increased segregation, a higher number of QTL are being detected in the F 2 transatlantic progeny Smolting in the F 2 s As mentioned, at the second measurement period when fish were almost two years old many were seen to be smolting, or near smolting, while others were clearly still parr due to the 26

35 presence of prominent parr marks. It is expected that within families there will be variability in the time of smolting, however this could also be attributed to the nature of the transatlantic cross. It has been observed that on average, the European subspecies smolts at different times than those from North America (Metcalfe and Thorpe, 1990). Therefore the segregation of North American and European alleles dealing with life history traits such as smoltification within the F 2 population could be causing this larger than average difference timing of the parr-smolt transformation. This observation is purely qualitative; therefore it would be interesting if future research focused on quantifying the proportion of smolts and non-smolts in comparison to each of the Mowi and Saint John s purebred strains. Environmental factors would also have to be considered, as smolting is heavily affected by such factors as photoperiod, food availability, tank density, and temperature (Thorpe et al., 1990). The remaining F 2 s are to be moved to saltwater in the fall of 2013, therefore at this time data could be obtained to investigate differences or similarities in the proportion and timing of smolting, or of smolt-transfer related mortalities between the F 2 population and each of the pure strains. 4.2 Length and Weight Length and weight in F 2 s in comparison to the literature It was decided to analyse length and weight as separate traits rather than combined into a condition factor (K), which gives a singular value based on the ratio between the two (calculated as (100 x body weight x full length -3, Boulding et al., (2008))). In this way direct comparisons between my results and those of condition factor in the transatlantic backcrosses cannot be made. However, as it was seen that a high number of linkage groups exhibiting significant QTL for length also mapped QTL for weight, and visa versa, I believe it is safe to draw general comparisons between the two studies in respect to length, weight and condition factor using the male-specific map. QTL for both early and late growth (condition factor) were mapped to Ssa02, Ssa07, and Ssa11 in the backcross population, with unique QTL for early growth on Ssa14, and Ssa04 and late growth on Ssa23 and Ssa26. Focusing on young parr, my results complement those of the backcrosses to some degree as QTL for length and weight at the first measurement period were mapped Ssa07, as well as a significant QTL for weight mapped to Ssa11 and Ssa02. Though more QTL were detected using the F 2 data than was found in the backcross population, Ssa14 did not contain any significant loci though it was of importance it the backcrosses. Results 27

36 for length and weight at the second measurement period revealed similarities to Boulding et al. (2008) where significant loci for both length and weight were mapped to Ssa07 and Ssa01/Ssa23, QTL for weight alone mapped to Ssa02, Ssa04, and Ssa26/Ssa28, and one QTL for length was located on Ssa11. Looking at weight independently, (which differed from results for growth on Ssa01/Ssa23 and Ssa26/Ssa28) QTL for early weight were found in both studies on Ssa02, Ssa07 (F 2 male map only), and Ssa11, while QTL significant in the backcrosses on Ssa14 and Ssa04 were not detected in the F 2 s. QTL for weight at measurement 2 with the male map were found in both studies on Ssa01/Ssa23, Ssa02, Ssa07, and Ssa26/Ssa28. While some linkage groups accounted for only one of the two traits, it is intriguing that length and weight, traits contributing to condition factor, were seen in similar locations to those in the backcrosses. Additionally, six of the eight linkage groups found by Reid et al. (2005) to be associated with body weight in North American Atlantic salmon were also mapped in the present study. QTL based on the male map were previously found on Ssa02, Ssa03, Ssa04, Ssa12 and Ssa21, which were also detected at both measurement periods in the F 2 s with the exception of Ssa03 and Ssa04 (that were only significant in older fish). Similarly, based on female recombination, Reid et al. (2008) located significant QTL for body weight on Ssa01, Ssa09, and Ssa15, where I found QTL on Ssa01 and Ssa09 at both measurement periods. Though these similarities are quite interesting, it is noteworthy that two of the largest QTL found by Reid et al. (2008) on Ssa03 and Ssa15 were not found using the F 2 population. Finally, a recent study by Gutierrez et al., (2012) mapped several significant QTL over 17 linkage groups (in fish months) for weight in the Mowi strain of European Atlantic salmon. While Gutierrez et al. (2012) performed 4 measurements, the 4 th measurement period was on fish 32 months old. Therefore, these fish were significantly older than my fish at their final measurement period (20 months) I only compared QTL to the first three measurements from the Gutierrez et al. (2012) study. Similarities were found between early weight on Ssa07 (F 2 male map only), Ssa09, Ssa11, and Ssa17 (F 2 female map only), which were detected in both populations. QTL at a later age were mapped on Ssa02, Ssa04, Ssa09, Ssa11, Ssa21 and Ssa24 (F 2 male map only) in both this study s transatlantic F 2 s and the Gutierrez et al. (2012) s Mowi strain. A summary of weight QTL comparison for the present study, Reid et al. (2005), Boulding et al. (2008), and Gutierrez et al. (2012) can be found in Table

37 4.2.2 Examination of candidate genes associated with length and weight Many QTL associated with length and with weight were mapped to chromosomes with known candidate genes important in growth and growth regulation. Growth hormone (GH), insulin-like growth factors (IGF1 and IGF2), as well as growth hormone receptors (GHR) are part of the growth hormone axis in teleost fish (Reinecke et al., 2005; Sakamoto et al., 1993). It is therefore interesting that I found QTL for length and weight at both measurement periods on Ssa01/Ssa23 and male-specific QTL for late (smolt) length and weight on Ssa24 on chromosomes known to contain the candidate gene GHR (Lien et al., 2011; Moghadam et al., 2007; Norman et al., 2012). In Atlantic salmon, GH1 and GH2 are located on Ssa03q and Ssa06q (Norman et al., 2012). I mapped female-specific QTL for early (parr) length and weight to 47cM and 44cM, respectively on Ssa03. I also mapped QTL for late length and weight both at 2cM on Ssa03 (female) and 33cM on Ssa06 (male). It is however less likely that GH is co-localizing with the QTL for late (near smolt) length in females as it was positioned at 2cM, and due to the approximate size of chromosome 3 being 104.2cM (Phillips et al., 2009), it would not be located on the q-arm. Moghadam et al. (2007) located candidate genes for IGF1 to Ssa07q and Ssa17p, and for IGF2 to Ssa10p and Ssa16p, all of which I found to contain at least one QTL for length or weight in the F 2 population. Using the female map, I found that a QTL for weight at the first measurement period positioned at 0cM co-localized with the candidate gene for IGF1 on the p- arm of Ssa17. Similarly, with the male-based map, I found QTL for length and weight at both measurement periods on Ssa07 at positions of 44cM (length 1), 42cM (weight 1), and 43cM (length2), were potentially colocalizing with IGF1. Though QTL for early length was also found on Ssa07 at a position of 20cM with the female map, due to the approximate size of Ssa07 being 104.2cM (Phillips et al., 2009), it is less likely that the length QTL in the F 2 s is actually colocalizing with IGF1 on the q-arm of Ssa07. QTL in the F 2 families may also be localizing with candidate genes for IGF2 on Ssa10p and Ssa16p; specifically QTL for weight at the first measurement period on Ssa10 (22cM with female map, 5cM with male map). I also found a 29

38 male-specific QTL for late length and weight on Ssa16, though this is less likely to co-localize with IGF2 because it was positioned at 67cM. 4.3 Homeologous Chromosomes The presence of QTL for two traits being on the same chromosome could be due to the linkage of separate QTL for each trait, or due to one QTL with pleiotropic effects accounting for both traits (Reid et al., 2005). I found that many QTL for both length and weight mapped to homeologous chromosomes. Many of the duplicated chromosome arms present in Atlantic salmon have been described, making it possible to ascertain the potential duplicated QTL positions (Danzmann et al., 2005; Danzmann et al., 2008; Lien et al., 2011). For example, I found that the homeologous chromosomes Ssa09/Ssa07, both contained mapped QTL for early length and parr mark number in females, as well as early and late length and weight, and parr mark number in males. Other possible duplicated QTL that were present on homeologs included 1) Ssa11/Ssa26 for weight at both measurement periods in females, and early weight, parr mark number, and skin contrast in males, 2) Ssa02/Ssa12 for male-specific weight at both measurement periods and parr mark number and contrast with the female map, and 3) Ssa06/Ssa09 for male-specific length and weight at the second measurement period. 4.4 Morphometric Traits The results of QTL analyses of the F 2 morphometric data revealed an immense number of significant loci across numerous linkage groups. In support of my initial hypothesis that QTL located in the Boulding et al. (2008) backcross study would map to similar locations in the F 2 population, it was found that the majority of QTL associated with morphometric landmarks in the backcrosses were also detected in the F 2 families. The major linkage groups containing most of the morphometric landmark QTL in the backcrosses were Ssa02, Ssa03, Ssa07, Ssa11, and Ssa23 (Boulding et al., 2008). I detected QTL corresponding to these linkage groups for the same traits, with the exceptions of 1) I did not detecting QTL for dorsal or for anal fin positions (DFx, DFy, AFx, AFy, respectively) on Ssa03, and 2) I did not detect QTL for the position of the operculum on Ssa09. Despite this, it is interesting that many of the landmarks had QTL in similar locations in both studies, and that both the backcrosses and F 2 s resulted in multiple QTL for each trait. This indicates that even specific morphological features are complex and perhaps controlled by polygenic genes. Though both studies detected many QTL associated with shape in 30

39 transatlantic, in accordance with my initial hypothesis, the F 2 data resulted in an overall greater number of significant QTL than were found in the backcrosses. The genetic basis of traits controlling shape is becoming an increasingly popular research topic. Despite this, no other studies, with the exception of Boulding et al., (2008), have looked at shape in Atlantic salmon. While it has been well documented that environmental factors can strongly influence shape in Atlantic salmon (Riddell and Leggett, 1981; Von Cramon-Taubade et al., 2005), studies based on other fish species have shown that shape also has a very strong genetic component. Zhang et al., (2012) investigated the genetic architecture of shape in the common carp (Cyprinus carpio) using SNPs and determined that it had a large genetic component. However, as in the present study, they could not determine if the effects were due to pleiotropy, or multiple loci controlling a single trait. A study utilizing microsatellites in sea bass controlled for environmental factors and still found that shape has a large genetic component (Costa et al., 2010). Albertson et al., (2003) investigated cichlid jaw shape, and showed that many variations in skeletal structure are inherited together, and were specifically associated with bone morphometric protein 4 (BMP4) transcript levels, suggesting that jaw shape has some degree of pleiotropy. Walker (2010) stresses the importance of considering all aspects of quantitative genetics, functional morphology and physiology, as well as functional ecology in order to understand the complex nature of shape. Therefore, the QTL associated with morphological traits in the F 2 population should be used as preliminary findings in order to provide insight into the genetic basis of shape in Atlantic salmon. 4.5 Accounting for Differences Between Families Significant differences were determined between families regarding weight, length, the number of parr marks, and their contrast to skin. Cross 1 was determined to be significantly shorter and to weigh less at both measurement periods compared to crosses 3 and 4. Similarly, cross 3 had significantly more parr marks and higher contrast than the remaining families. While I cannot conclusively explain these differences, I propose a couple of possible hypotheses, as well as rule out other possibilities. The first possible explanation that comes to mind is environmental effects. As all traits do have an environmental component, it is possible to see such variations within families. However, in this study, up until the time of the first 31

40 measurement, the three families were split up into six tanks, two for each family with approximately equal densities. Furthermore, once fish were PIT-tagged and were large enough to move into two indoor tanks, all fish were assigned to tanks randomly, mixing families. In this respect, tank effects are unlikely to be the cause of the phenotypic variation. Feed amount could also be a factor causing variation in phenotypes, specifically size, however fish are fed in a random manner by hand in order to limit aggression from monopolizing a feeder; therefore it is unlikely that aggressive behaviour (or calmer behaviour in the case of cross 1) is the cause of differences in size between families. While genetic differences among subspecies have been emphasized, heritable genetic variation in traits also occur within each subspecies. Therefore one possible hypothesis could be that the parents of cross 1 are genetically smaller and lighter than the parents of crosses 3 and 4, and that the parents of cross 3 happen to possess many alleles for a high number of parr marks and light skin. All traits have been shown to be heritable (with the exception of skin contrast), therefore progeny of these two crosses would be inheriting the parental alleles associated with the specific traits, leading to differences between families. Another possible hypothesis relates to the potential uneven Mendelian segregation of North American and European alleles arising from different number of chromosomes. Especially in relation to parr mark number and contrast, the North American and European subspecies differ morphologically (Boulding et al., 2008). Therefore a cross that inherits an uneven amount of alleles from one subspecies over the other, may display significantly different phenotypes. 4.6 Conclusions and Implications Overall, my hypotheses and predictions were upheld with some exceptions. While many of the QTL found in this study were not in similar locations to those of Boulding et al. (2008), I believe they can be attributed to the differences between F 2 and backcross transatlantic populations. In relation to my second hypotheses that segregation of alleles from the two subspecies would result in more detectable QTL than previous studies, it is possible that the QTL found in the backcross populations have lower effects in the F 2 s and were therefore not of high significance. QTL analyses were done at the experiment-wide level only, therefore QTL that were not similar between the two studies could be significant at the chromosome-wide level, however were not reported. 32

41 While higher densities of SNPs should be used in future studies, the number used for this F 2 population allowed for more accurate estimations of the locations of QTL in comparison to those of the backcrosses. The added multiplex allowed for all linkage groups to contain informative SNPs, as well as giving relatively good coverage along the length of the chromosome. The use of F 2 crosses allowed for more precise mapping of QTL associated with length, weight, parr mark number, parr mark contrast, and shape. Future studies, perhaps on F 3 crosses, should focus on higher-resolution mapping in order to determine if QTL are few with large effects, or many with small effects that cannot be distinguished, especially in males due to their lower recombination rates (Moen et al., 2004). When considering the potential use of QTL for marker-assisted selection (MAS) in Atlantic salmon, the loci detected in the F 2 s may not be directly applicable to purebred strains. It does however provide insight into possible fixed loci for economically important traits such as length and weight. Similarly, this study shows the large genetic control for parr marks, which may aid in future conservation efforts. Being able to introduce Atlantic salmon into the wild that are well suited to their environment, specifically having parr mark colouration which closely matches that of its substrate, can increase overall survival through reduced predation. Finally, possibly one of the most interesting findings of this study is that North American and European Atlantic salmon can produce viable F 2 progeny, despite their differences in karyotype. This alone is extremely interesting, especially in regard to the presence of fused chromosomes in the North American subspecies. The introduction of European aquaculture strains into Canada has been discussed, though the possibility of European escapees interbreeding with the native population has been of major concern. This study shows that the two subspecies are capable of breeding, however it is still unknown if the F 2 s are able to produce viable F 3 s. Therefore, it would be incredibly interesting is future work involved karyotyping the F 2 s in order to see the deviations in karyotypes from both native lines, especially on the chromosomes (01/23, 08/29 and 26/28) that are fused in the North American subspecies. 5.0 References Agriculture and Agri-Food Canada., Fish and seafood facts sheet: farmed salmon. [Accessed 2013 January 22] 33

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48 6.0 Tables Table 1 Description of landmark positions and x/y coordinate names Description Horizontal coordinate name Vertical coordinate name Tip of snout SNx SNy Anterior insertion of dorsal fin DFx DFy Dorsal insertion of caudal fin CFx CFy End of caudal peduncle at the lateral line CPx CPy Fork of the caudal fin FKx FKy Anterior insertion of anal fin AFx AFy Anterior insertion of pelvic fin PLFx PLFy Most anterior point of pectoral fin AnPCFx AnPCFy Most ventral and posterior insertion of pectoral fin vpcfx vpcfy Most anterior and dorsal insertion of pectoral fin dpcfx dpcfx Point where operculum meets ventral body line OPx OPy Most anterior point of pupil PUx PUy 40

49 Table 2 Summary of means for phenotypic traits in all families of F 2 transatlantic salmon progeny. No. Individuals Measurement 1 No. Individuals Measurement 2 c Length1 (cm) a Weight1 (g) a Length2 (cm) b Weight2 (g) b No. Parr Marks a Skin Contrast a Cross ± ± ± ± ± ±0.02 Cross ± ± ± ± ± ±0.02 Cross 4 c 347 N/A 9.38± ±0.22 N/A N/A 8.79± ±0.03 a Measurement 1 took place November 15 th -25 th, b Measurement 2 took place November 28 th -30 th, c Cross 4 were euthanized at the end of the first measurement period. 41

50 Table 3 Linkage groups associated with significant QTL for length (length1) and weight (weight1) at first measurement period in three F 2 transatlantic salmon families using map based on female meioses. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 length ** ± /23 17/18 weight ** ± length ** ± weight ** ± length * ± weight ** ± length * ± length ** ± weight ** ± weight ** ± weight ** ± weight ** ± length ** ± weight ** ± length ** ± weight ** ± /28 21/33 length ** ± /28 21/33 weight ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =1047). f Proportion of phenotypic variance accounted for by QTL 42

51 Table 4 Linkage groups associated with significant QTL for length (length1) and weight (weight1) at first measurement period in three F 2 transatlantic salmon families using map based on male meioses. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 length ** ± /23 17/18 weight ** ± weight ** ± length ** ± weight ** ± length ** ± weight ** ± weight ** ± weight ** ± weight * ± length ** ± weight ** ± /28 21/33 weight ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =1047). f Proportion of phenotypic variance accounted for by QTL 43

52 Table 5 Linkage groups associated with significant QTL for length (length2) and weight (weight2) at second measurement period in two F 2 transatlantic salmon families using map based on female meiosis. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 weight ** ± /23 17/18 length ** ± weight ** ± length ** ± weight ** ± length ** ± weight * ± weight ** ± length * ± weight ** ± length ** ± weight ** ± length ** ± /28 21/33 weight ** ± /28 21/33 length ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =499). f Proportion of phenotypic variance accounted for by QTL 44

53 Table 6 Linkage groups associated with significant QTL for length (length2) and weight (weight2) at second measurement period in two F 2 transatlantic salmon families using map based on male meioses. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 weight ** ± /23 17/18 length ** ± weight * ± weight * ± weight ** ± length ** ± weight ** ± length * ± weight ** ± length ** ± length ** ± weight ** ± length * ± weight2 0 * 24.4** ± length2 0 * 16.20** ± weight ** ± length ** ± weight ** ± length * ± /28 21/33 weight * ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =499). f Proportion of phenotypic variance accounted for by QTL * Position based on attachment with Ssa19 on linkage map for computational facility. 45

54 Table 7 Linkage groups with significant QTL for parr mark number (number) and relative contrast of skin and parr mark colouration (contrast) in three F 2 transatlantic salmon families using map based on female meioses. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 number ** ± /23 17/18 contrast ** ± number ** ± contrast ** ± number ** ± contrast ** ± number ** ± contrast ** ± number * ,75± number ** ± contrast * ± number ** ± contrast * ± number ** ± contrast * ± number ** ± contrast ** ± number ** ± contrast ** ± number ** ± number 0 * 42.76** ± contrast 0 * 24.03** ± number ** ± contrast ** ± number ** ± contrast ** ± number ** ± contrast ** ± /28 21/33 number ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =1046). f Proportion of phenotypic variance accounted for by QTL * Position based on attachment with Ssa19 on linkage map for computational facility. 46

55 Table 8 Linkage groups with significant QTL for parr mark number (number) and relative contrast of skin and parr mark colouration (contrast) in three F 2 transatlantic salmon families using map based on male meioses. Chr a LG b Trait Pos(cM) c F d df Mean e PEV f 1/23 17/18 number ** ± /23 17/18 contrast ** ± number ** ± number ** ± contrast ** ± contrast * ± number ** ± contrast * ± number * ± number * ± contrast * ± number * ± number ** ± contrast ** ± number ** ± contrast ** ± number * ± number 0 * 42.76** ± contrast 0 * 24.05** ± number ** ± contrast ** ± number ** ± contrast ** number ** ± contrast ** ± /28 21/33 number ** ± /28 21/33 contrast ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Position on chromosome d F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. e Mean and standard error of trait mean (N max =1046). f Proportion of phenotypic variance accounted for by QTL * Position based on attachment with Ssa19 on linkage map for computational facility. 47

56 Table 9 Linkage groups with significant QTL for 11 landmarks at first measurement period in three F 2 transatlantic salmon families using map based on female meioses. Chr a LG b Trait c Pos(cM) d F e df Mean f PEV g 1/23 17/18 SNy ** ± /23 17/18 DFx ** ± /23 17/18 DFy ** ± /23 17/18 CFx ** ± /23 17/18 CPx ** ± /23 17/18 CPy ** ± /23 17/18 FKx ** ± /23 17/18 FKy ** ± /23 17/18 AFx ** ± /23 17/18 AFy ** ± /23 17/18 PLFy ** ± /23 17/18 dpcfx ** ± /23 17/18 OPx ** ± /23 17/18 PUy ** ± SNx ** ± SNy ** ± DFx ** ± DFy ** ± CFx ** ± CFy ** ± CPx ** ± CPy ** ± FKx ** ± FKy ** ± AFx ** ± AFy ** ± PLFy ** ± vpcfx ** ± dpcfx ** ± dpcfy ** ± PUy ** ± SNy ** ± DFx ** ± DFy ** ± CPy ** ± FKy ** ± AFy ** ± PLFy ** ± dpcfx ** ± PUy ** ± SNy ** ± DFx ** ± DFy ** ±

57 4 28 CPy ** ± FKy ** ± AFy ** ± PLFy ** ± PUy ** ± dpcfx ** ± DFx ** ± DFy ** ± CPx ** ± FKx ** ± dpcfx ** ± AFx ** ± dpcfy ** ± DFx ** ± DFy ** ± CPx ** ± PLFy ** ± DFx ** ± DFy ** ± dpcfx ** ± DFx ** ± DFy ** ± CPy ** ± PLFy ** ± DFx ** ± DFy ** ± CPx ** ± FKx ** ± dpcfx ** ± SNy 0 * 14.04** ± DFx 0 * 37.05** ± DFy 0 * 39.3** ± CPx 0 * 8.39** ± CPy 0 * 28.65** ± FKy 0 * 14.45** ± AFy 0 * 17.27** ± PLFy 0 * 26.44** ± PUy 0 * 20.89** ± DFx ** ± DFy ** ± AFx ** ± dpcfx ** ± SNy ** ± DFx ** ± DFy ** ± CFx ** ±

58 21 14 CFy ** ± CPx ** ± CPy ** ± FKx ** ± FKy ** ± AFx ** ± AFy ** ± PLFy ** ± vpcfx 2 9.6** ± dpcfx ** ± OPx ** ± PUy ** ± DFx ** ± DFy ** ± PLFy ** ± DFx ** ± DFy ** ± CPx ** ± dpcfx ** ± /28 21/33 DFx ** ± /28 21/33 DFy ** ± /28 21/33 CFx ** ± /28 21/33 CPx ** ± /28 21/33 FKx ** ± /28 21/33 AFx ** ± /28 21/33 dpcfx ** ± /28 21/33 dpcfy ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Morphometric landmark positions, see text for description of landmark d Position on chromosome e F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. f Mean and standard error of trait mean (N max =1046). g Proportion of phenotypic variance accounted for by QTL * Position based on attachment with Ssa19 on linkage map for computational facility. 50

59 Table 10 Linkage groups with significant QTL for 11 landmarks at first measurement period in three F 2 transatlantic salmon families using map based on male meioses. Chr a LG b Trait c Pos(cM) d F e df Mean f PEV g 1/23 17/18 SNy ** ± /23 17/18 DFx ** ± /23 17/18 DFy ** ± /23 17/18 CFx ** ± /23 17/18 CPx ** ± /23 17/18 CPy ** ± /23 17/18 FKx ** ± /23 17/18 FKy ** ± /23 17/18 AFx ** ± /23 17/18 PLFy ** ± /23 17/18 vpcfx ** ± /23 17/18 dpcfx ** ± /23 17/18 PUy ** ± SNy ** ± DFx ** ± DFy ** ± CFy ** ± CPx ** ± CPy ** ± FKy ** ± AFy ** ± PLFy ** ± vpcfx ** ± PUy ** ± dpcfx ** ± SNy ** ± DFx ** ± DFy ** ± CPy ** ± FKy ** ± PLFy ** ± PUy ** ± DFx ** ± DFy ** ± CPy ** ± PLFy ** ± DFx ** ± CFx ** ± dpcfx ** ± OPx ** ± dpcfx ** ± dpcfy ** ± DFx ** ±

60 11 9 DFy ** ± PLFy ** ± DFx ** ± FKx ** ± dpcfx ** ± DFx ** ± DFy ** ± CPy ** ± PLFy ** ± SNy 0 * 14.05** ± DFx 0 * 37.08** ± DFy 0 * 39.33** ± CPx 0 * 8.4** ± CPy 0 * 28.66** ± FKy 0 * 14.46** ± AFy 0 * 17.28** ± PLFy 0 * 26.45** ± PUy 0 * 20.9** ± CFx 0 * 10.28** ± dpcfx ** ± DFx ** ± DFy ** ± CPy ** ± DFx ** ± DFy ** ± /28 21/33 SNy ** ± /28 21/33 DFx ** ± /28 21/33 DFy ** ± /28 21/33 CPy ** ± /28 21/33 PLFy ** ± /28 21/33 PUy ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Morphometric landmark positions, see text for description of landmark d Position on chromosome e F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. f Mean and standard error of trait mean (N max =1046). g Proportion of phenotypic variance accounted for by QTL * Position based on attachment with Ssa19 on linkage map for computational facility. 52

61 Table 11 Linkage groups with significant QTL for 11 landmarks at second measurement period in three F 2 transatlantic salmon families using map based on female meioses. Chr a LG b Trait c Pos(cM) d F e df Mean f PEV g 1/23 17/18 SNx ** ± /23 17/18 DFy ** ± /23 17/18 CPx ** ± /23 17/18 FKy ** ± /23 17/18 AFy ** ± /23 17/18 PLFx ** ± /23 17/18 vpcfx ** ± /23 17/18 vpcfy ** ± /23 17/18 dpcfx ** ± /23 17/18 dpcfy ** ± /23 17/18 OPy ** ± /23 17/18 PUx ** ± vpcfx ** ± vpcfy ** ± SNx ** ± DFy ** ± CPx ** ± FKx ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± dpcfx ** ± dpcfy ** ± OPy ** ± PUx ** ± SNx ** ± DFy ** ± CPx ** ± FKx ** ± vpcfx ** ± vpcfy ** ± dpcfy ** ± PUx ** ± vpcfy ** ± DFy ** ± vpcfy ** ± vpcfx ** ± vpcfy ** ± vpcfy ** ± vpcfx ** ± vpcfy ** ± PUx ** ± CPx ** ±

62 13 5 vpcfx ** ± vpcfy ** ± SNx ** ± DFy ** ± CPx ** ± FKx ** ± FKy ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± dpcfx ** ± dpcfy ** ± OPy ** ± PUx ** ± vpcfx ** ± vpcfy ** ± PUx ** ± SNx ** ± DFy ** ± FKx 0 8.1** ± AFy ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± PUx ** ± /28 21/33 SNx ** ± /28 21/33 DFy ** ± /28 21/33 CPx ** ± /28 21/33 FKx ** ± /28 21/33 FKy ** ± /28 21/33 AFy ** ± /28 21/33 PLFx ** ± /28 21/33 vpcfx ** ± /28 21/33 vpcfy ** ± /28 21/33 dpcfy ** ± /28 21/33 OPy ** ± /28 21/33 PUx ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Morphometric landmark positions, see text for description of landmark d Position on chromosome e F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. f Mean and standard error of trait mean (N max =499). g Proportion of phenotypic variance accounted for by QTL 54

63 Table 12 Linkage groups with significant QTL for 11 landmarks at second measurement period in three F 2 transatlantic salmon families using map based on male meioses. Chr a LG b Trait c Pos(cM) d F e df Mean f PEV g 1/23 17/18 SNx ** ± /23 17/18 CPx ** ± /23 17/18 DFy ** ± /23 17/18 FKx ** ± /23 17/18 FKy ** ± /23 17/18 AFy ** ± /23 17/18 PLFx ** ± /23 17/18 vpcfx ** ± /23 17/18 vpcfy ** ± /23 17/18 dpcfx ** ± /23 17/18 dpcfy ** ± /23 17/18 PUx ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± SNx ** ± DFy ** ± CPx ** ± vpcfx ** ± vpcfy ** ± dpcfy ** ± OPy ** ± PUx ** ± SNx ** ± DFy ** ± CPx ** ± FKx ** ± FKy ** ± AFy 43 8** ± PLFx ** ± vpcfx ** ± vpcfy ** ± dpcfx ** ± dpcfy ** ± PUx ** ± SNx ** ± vpcfx ** ± vpcfy ** ± vpcfx 5 6.8** ± SNx ** ± vpcfx ** ± vpcfy ** ± PUx ** ±

64 13 5 CPx ** ± vpcfx 30 10** ± vpcfy ** ± SNx ** ± DFy ** ± CPx ** ± FKx ** ± FKy ** ± AFy ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± dpcfx ** ± dpcfy ** ± OPy ** ± PUx ** ± SNx ** ± DFy ** ± CPx ** ± vpcfx ** ± vpcfy ** ± dpcfy ** ± PUx ** ± SNx ** ± DFy ** ± FKx ** ± AFy ** ± PLFx ** ± vpcfx ** ± vpcfy ** ± PUx ** ± /28 21/33 vpcfy ** ± a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 c Morphometric landmark positions, see text for description of landmark d Position on chromosome e F-test with experiment-wide permutation (* indicates significance at P<0.05, ** indicates significance at P<0.01) using GridQTL. f Mean and standard error of trait mean (N max =499). g Proportion of phenotypic variance accounted for by QTL 56

65 Table 13 QTL for weight as described by Reid et al. (2005), Boulding et al. (2008), and Gutierrez et al. (2012) in comparison to the present study. Chr LG (2005) (2008) (2012) This study a b Reid et al. Boulding et al., Gutierrez et al., 1 17 W1 W1*, W2* 2 1 W1 W1, W2 W2 W1, W W1 W1, W W1 W1 W1, W2 W W1, W2 6 4 W W1, W2 W1 W W2, W W1 W1, W2 W1, W W2 W W1, W2 W1, W2 W1, W W W W W1 W1, W W W1 W W1, W W W1 W2 W1, W W W2 W1*, W2* 24 7 W1, W2 W W W2 W1*, W2* W1*, W2* W1, W2 a Atlantic salmon Ssa chromosome number Phillips et al., 2009 b Linkage group using numbering system in Phillips et al., 2009 W1 = QTL detected from measurements on fish 7-12 months of age, W2 = QTL detected from measurements on fish months of age. *indicates QTL detected on chromosomes fused in North American subspecies, QTL could therefore be present on either chromosome in the fusion depending on orientation and segregation. 57

66 7.0 Figures Figure 1 Atlantic salmon life cycle. Atlantic Salmon Federation, asf.ca. 58

67 a) b) Figure 2 a) Landmarks for morphometric digitizing: 1. Tip of snout (SN), 2. Anterior insertion of dorsal fin (DF), 3. Dorsal insertion of caudal fin (CF), 4. End of caudal peduncle at the lateral line (CP), 5. Fork of caudal fin (FK), 6. Anterior insertion of anal fin (AF), 7. Anterior insertion of pelvic fin (PLF), 8. Most anterior point of pectoral fin (AnPCF), 9. Most ventral and posterior insertion of pectoral fin (vpcf), 10. Most anterior and dorsal insertion of pectoral fin (dpcf), 11. Point where the operculum meets the ventral body line (OP), 12. Most anterior point of pupil (PU). b) Actual photograph of fish that was digitized at second measurement period. 59

68 a) b) c) Figure 3 Visualization of shape variation in relation to centroid size. Landmark numbers correspond to: 1. Tip of snout (SN), 2. Dorsal Fin (DF), 3. Caudal fin (CF), 4. Caudal peduncle (CP), 5. Fork of caudal fin (FK), 6. Anal fin (AF), 7. Pelvic fin (PLF), 9. Ventral insertion of pectoral fin (vpcf), 10. Dorsal insertion of pectoral fin (dpcf), 11. Operculum (OP), 12. Pupil (PU) a) Average centroid size b) Vector displacement (arrows) seen in fish of smallest composite size c) Vector displacement (arrows) seen in fish of largest composite size. 60

69 a) b) Figure 4 a) Measurements for colour contrast of skin to parr marks. Blue boxes indicate 5x5 pixel area for brightness measurement on parr marks, red boxes indicated 5x5 pixel area for skin measurement. b) Actual photograph of parr used for contrast measurements. 61

70 Figure 5 Distribution of length (cm) at first measurement over all three F 2 families. Measurements were obtained at SABS November 15 th -25 th, n=1047, mean=9.04±0.04, skewness=0.02, kurtosis=

71 Figure 6 Distribution of weight (g) at first measurement for all three F 2 crosses. Measurements were obtained at SABS November 15 th -25 th, n= 1047, mean=8.74±0.12, skewness=0.55, kurtosis=

72 Figure 7 Distribution of length at second measurement of remaining fish in F 2 crosses 1 and 3. Measurements were performed at SABS November 28 th -30 th, n=499, mean=19.18±0.14, skewness=0.18, kurtosis=

73 Figure 8 Distribution of weight (g) at second measurement for F 2 crosses 1 and 3. Measurements were performed at SABS November 28 th -30 th, n=499, mean=95.10±1.73, skewness=0.69, kurtosis=

74 Figure 9 Distribution of the number of parr marks per individual over all three F 2 crosses. Measurements were obtained at SABS November 15 th -25 th, n=1046; mean=8.99±0.03, skewness=0.19, kurtosis=

75 Figure 10 Distribution of the relative contrast of skin colouration to parr marks over all three F 2 families. The distribution is positively skewed, with the majority of individuals having low contrast values. Measurements were obtained at SABS November 15 th -25 th, n=1046, mean=1.88±0.01, skewness=2.56, kurtosis=

76 Figure 11 Examples of selected colour morphologies from second measurement period. a) Parr with few very bright red spots. b) Parr with many less vivid red spots. c) Smolt with very bright sliver colouration. d) Smolt with silver/blue colouration. e) Parr without red spots. 68