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Males and females often differ in their fitness optima for shared traits that have a shared genetic basis, leading to sexual conflict. Morphologically differentiated sex chromosomes can resolve this conflict and protect sexually antagonistic variation, but they accumulate deleterious mutations. However, how sexual conflict is resolved in species that lack differentiated sex chromosomes is largely unknown. Here we present a chromosome-anchored genome assembly for rainbow trout (Oncorhynchus mykiss) and characterize a 55-Mb double-inversion supergene that mediates sex-specific migratory tendency through sex-dependent dominance reversal, an alternative mechanism for resolving sexual conflict. The double inversion contains key photosensory, circadian rhythm, adiposity and sex-related genes and displays a latitudinal frequency cline, indicating environmentally dependent selection. Our results show sex-dependent dominance reversal across a large autosomal supergene, a mechanism for sexual conflict resolution capable of protecting sexually antagonistic variation while avoiding the homozygous lethality and deleterious mutations associated with typical heteromorphic sex chromosomes.

Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is shown through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13. USDA Agricultural Research Service | Ref. 8082-31000-012 Agriculture and Food Research Initiative Competitive | Ref. 2015-07185 USDA National Institute of Food and Agriculture

Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2N=64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.Article SummaryA de-novo genome assembly was generated for the Arlee homozygous line of rainbow trout to enable identification and characterization of genome variants towards developing a rainbow trout pan-genome reference. The new assembly was generated using the PacBio sequencing technology and scaffolding with Hi-C contact maps and Bionano optical mapping. A contiguous genome assembly was obtained, with the contig and scaffold N50 over 15.6 Mb and 39 Mb, respectively, and 95% of the assembly in chromosome sequences. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes.

Cold-water dependent rainbow trout (Oncorhynchus mykiss) inhabit a considerably diverse geographic range extending from Alaska to southern California, where adjustments in growth and swim performance in response to temperature may be present. Here we measured four clonal lines of rainbow trout for growth and sprint speed, at two different constant temperatures (10 or 18 °C), across 12 weeks. The objective was to characterize temperature responses among different genotypes originating from Alaska, Oregon, and two hatchery populations; we expected that the lines would respond in a way that implicated their diversity in geographic origin and domestication history. We observed all fish achieve the greatest growth and body size and fastest sprint speed at 18 °C, regardless of origin; substantial variation in growth and swim performance among and within line and temperature was also observed. Surprisingly, the Swanson line from Alaska exhibited very rapid growth at the warmer temperature, which may support the countergradient variation in growth hypothesis. The diversity of responses reported here illustrates the complex nature of the relationship among temperature, growth, and performance and provide groundwork for future genetic analyses utilizing clonal lines of rainbow trout.

Traits with different fitness optima in males and females cause sexual conflict when they have a shared genetic basis. Heteromorphic sex chromosomes can resolve this conflict and protect sexually antagonistic polymorphisms but accumulate deleterious mutations. However, many taxa lack differentiated sex chromosomes, and how sexual conflict is resolved in these species is largely unknown. Here we present a chromosome-anchored genome assembly for rainbow trout (Oncorhynchus mykiss) and characterize a 56 Mb double-inversion supergene that mediates sex-specific migration through sex-dependent dominance, a mechanism that reduces sexual conflict. The double-inversion contains key photosensory, circadian rhythm, adiposity, and sexual differentiation genes and displays frequency clines associated with latitude and temperature, revealing environmental dependence. Our results constitute the first example of sex-dependent dominance across a large autosomal supergene, a novel mechanism for sexual conflict resolution capable of protecting polygenic sexually antagonistic variation while avoiding the homozygous lethality and deleterious mutation load of heteromorphic sex chromosomes.

Organisms can adapt to local environmental conditions as a plastic response or become adapted through natural selection on genetic variation. The ability to adapt to increased water temperatures will be of paramount importance for many fish species as the climate continues to warm and water resources become limited. Because increased water temperatures will reduce the dissolved oxygen available for fish, we hypothesized that adaptation to low oxygen environments would involve improved respiration through oxidative phosphorylation (OXPHOS). To test this hypothesis, we subjected individuals from two ecologically divergent populations of inland (redband) rainbow trout (Oncorhynchus mykiss gairdneri) with historically different temperature regimes (desert and montane) and their F1 progeny to diel cycles of temperature stress and then examined gene expression data for 80 nuclear- and mitochondrial-encoded OXPHOS subunits that participate in respiration. Of the 80 transcripts, 7 showed ≥ 2-fold difference in expression levels in gill tissue from desert fish under heat stress whereas the montane fish had none and the F1 only had one differentially expressed gene. A structural analysis of the proteins encoded by those genes suggests that the response could coordinate the formation of supercomplexes and oligomers. Supercomplexes may increase the efficiency of respiration because complexes I, III, and IV are brought into close proximity and oligomerization of complex V alters the macrostructure of mitochondria to improve respiration. Significant differences in gene expression patterns in response to heat stress in a common environment indicate that the response was not due to plasticity but had a genetic basis.

Fish domestication is an evolutionary process arising in captivity through genetic and developmental mechanisms, producing organisms performing more poorly than wild conspecifics in the natural environment. Culture conditions could be suboptimal for fish at particular life cycle stages, presenting environmental disturbances leading to developmental instability. The limited size of captive lots, moreover, can result in the loss of genetic variation, and the resulting homozygosity (as well as hybridization and mutation) could have strong harmful effects on developmental stability. Rainbow trout are the most widely-cultured species in Europe and North America, having been in culture for more than a century. Prolonged artificial selection for desired traits and incidental effects of domestication has led to the development of a ‘farmed type’. Fluctuating asymmetry, variations in meristic counts, and skeletal anomalies were examined in several rainbow trout captive and wild clonal lines as indicators of developmental instability. Differences in developmental stability were identified among lines and correlated with different degrees of exposure to captivity. Some relationship between meristic counts and domestication level was found in the present study, with the number of vertebrae and of dorsal pterygiophores and rays being the strongest predictors of the domestication level. However, the occurrence of skeletal anomalies and fluctuating asymmetry were apparently not related to the level of exposure to captivity. The findings of the present study will facilitate the selection of clonal lines with divergent phenotypes for subsequent quantitative trait loci analyses aimed at identifying genome regions linked with morpho-anatomical and physiological adaptive responses to captivity.

Domestication causes behavior and brain size changes in many species. We addressed three questions using clonal rainbow trout lines: What are the mirror-elicited aggressive tendencies in lines with varying degrees of domestication? How does brain size relate to genotype and domestication level? Finally, is there a relationship between aggressive behavior and brain size? Clonal lines, although sampling a limited subset of the species variation, provide us with a reproducible experimental system with which we can develop hypotheses for further research. We performed principal component analyses on 12 continuous behavior and brain/body size variables and one discrete behavioral variable ("yawn") and detected several aggression syndromes. Two behaviors, "freeze" and "escape", associated with high domestication; "display" and "yawn" behavior associated with wild lines and "swim against the mirror" behavior associated with semi-wild and domestic lines. Two brain size traits, total brain and olfactory volume, were significantly related to domestication level when taking total body size into account, with domesticated lines having larger total brain volume and olfactory regions. The aggression syndromes identified indicate that future QTL mapping studies on domestication-related traits would likely be fruitful.

The Columbia River and its tributaries provide essential spawning and rearing habitat for many salmonid species, including Chinook salmon (Oncorhynchus tshawytscha). Chinook salmon were historically abundant throughout the basin and Native Americans in the region relied heavily on these fish for thousands of years. Following the arrival of Europeans in the 1800s, salmon in the basin experienced broad declines linked to overfishing, water diversion projects, habitat destruction, connectivity reduction, introgression with hatchery-origin fish, and hydropower development. Despite historical abundance, many native salmonids are now at risk of extinction. Research and management related to Chinook salmon is usually explored under what are termed "the four H's": habitat, harvest, hatcheries, and hydropower; here we explore a fifth H, history. Patterns of prehistoric and contemporary mitochondrial DNA variation from Chinook salmon were analyzed to characterize and compare population genetic diversity prior to recent alterations and, thus, elucidate a deeper history for this species. A total of 346 ancient and 366 contemporary samples were processed during this study. Species was determined for 130 of the ancient samples and control region haplotypes of 84 of these were sequenced. Diversity estimates from these 84 ancient Chinook salmon were compared to 379 contemporary samples. Our analysis provides the first direct measure of reduced genetic diversity for Chinook salmon from the ancient to the contemporary period, as measured both in direct loss of mitochondrial haplotypes and reductions in haplotype and nucleotide diversity. However, these losses do not appear equal across the basin, with higher losses of diversity in the mid-Columbia than in the Snake subbasin. The results are unexpected, as the two groups were predicted to share a common history as parts of the larger Columbia River Basin, and instead indicate that Chinook salmon in these subbasins may have divergent demographic histories.

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout (Oncorhynchus mykiss), SNP discovery has been previously done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL) and RNA sequencing. Recently we have performed high coverage whole genome resequencing with 61 unrelated samples, representing a wide range of rainbow trout and steelhead populations, with 49 new samples added to 12 aquaculture samples from AquaGen (Norway) that we previously used for SNP discovery. Of the 49 new samples, 11 were double-haploid lines from Washington State University (WSU) and 38 represented wild and hatchery populations from a wide range of geographic distribution and with divergent migratory phenotypes. We then mapped the sequences to the new rainbow trout reference genome assembly (GCA_002163495.1) which is based on the Swanson YY doubled haploid line. Variant calling was conducted with FreeBayes and SAMtools mpileup, followed by filtering of SNPs based on quality score, sequence complexity, read depth on the locus, and number of genotyped samples. Results from the two variant calling programs were compared and genotypes of the double haploid samples were used for detecting and filtering putative paralogous sequence variants (PSVs) and multi-sequence variants (MSVs). Overall, 30,302,087 SNPs were identified on the rainbow trout genome 29 chromosomes and 1,139,018 on unplaced scaffolds, with 4,042,723 SNPs having high minor allele frequency (MAF > 0.25). The average SNP density on the chromosomes was one SNP per 64 bp, or 15.6 SNPs per 1 kb. Results from the phylogenetic analysis that we conducted indicate that the SNP markers contain enough population-specific polymorphisms for recovering population relationships despite the small sample size used. Intra-Population polymorphism assessment revealed high level of polymorphism and heterozygosity within each population. We also provide functional annotation based on the genome position of each SNP and evaluate the use of clonal lines for filtering of PSVs and MSVs. These SNPs form a new database, which provides an important resource for a new high density SNP array design and for other SNP genotyping platforms used for genetic and genomics studies of this iconic salmonid fish species.

Yersinia ruckeri is a well-established bacterial pathogen for many salmonid species, against which a formalin-killed bacterin vaccine has been effective in reducing disease outbreaks. Previous studies have reported conflicting results about the protective value of the systemic humoral response to Y. ruckeri vaccination. Here we directly demonstrate that plasma contains the long-term protective component elicited by both immersion and intraperitoneal injection vaccination of rainbow trout. A total of 0.5 μL of plasma from vaccinated fish provided almost complete protection against experimental challenge. Conversely, the cells obtained from peripheral blood conferred little or no protection in naive recipients. The protective component of immune sera was IgM based on size exclusion chromatography and recognition by monoclonal antibody Warr 1–14. Immune plasma generated against a Y. ruckeri biotype 1 strain protected equally against challenges with Y. ruckeri biotype 1 and 2 strains. These results illustrate the importance of the humoral IgM response against Y. ruckeri and the use of doubled haploid rainbow trout ( Oncorhynchus mykiss ) and transfer of plasma/serum and cells into F1 outcross progeny as a model system for dissection of the mechanism(s) of vaccine-induced protection.

Development rate has important implications for individual fitness and physiology. In salmonid fishes, development rate cor relates with many traits later in life, including life-history diversity, growth, and age and size at sexual maturation. In rainbow trout (Oncorhynchus mykiss), a quantitative trait locus for embryonic development rate has been detected on chromosome 5 across populations. However, few candidate genes have been identified within this region. In this study, we use gene map ping, gene expression, and quantitative genetic methods to further identify the genetic basis of embryonic developmental rate in O. mykiss. Among the genes located in the region of the major development rate quantitative trait locus (GHR1, Clock1a, Myd118-1, and their paralogs), all were expressed early in embryonic development (fertilization through hatch), but none were differentially expressed between individuals with the fast- or slow-developing alleles for a major embryonic development rate quantitative trait locus. In a follow-up study of migratory and resident rainbow trout from natural populations in Alaska, we found significant additive variation in development rate and, moreover, found associations between development rate and allelic variation in all 3 candidate genes within the quantitative trait locus for embryonic development. The mapping of these genes to this region and associations in multiple populations provide positional candidates for further study of their roles in growth, development, and life-history diversity in this model salmonid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Domestication selection combined with inadvertent selection for high growth rate and large body size are common in hatchery salmonids. However, evolutionary trade-offs of various fitness components with body size and domestication are likely. This study explores potential trade-offs between growth and sprint swim performance among five clonal lines of rainbow trout ( Oncorhynchus mykiss ), varying in the number of generations under domestication. We hypothesized that clonal lines of trout derived from highly domesticated populations would have higher growth rates but slower sprint swimming performance relative to clonal lines derived from less-domesticated populations. Individuals from each of the five lines were repeatedly measured for body size and sprint swimming performance over 10–15 weeks. Our findings indicate significant differences in body size and sprint swimming performances between the less-domesticated and the more-domesticated lines, with more-domesticated lines being larger but poorer swimmers. Implications for the survival and reproduction of hatchery salmonids released into the wild are explored.

Natural variation in the antioxidant-enzyme SOD-1 (superoxide dismutase) is known to alter the impacts of oxidative damage at both the cellular and organismal levels. Using three homozygous clonal lines of rainbow trout [Hot Creek (n=30), Arlee (n=21), and Swanson (n=10)], which differ for single nucleotide polymorphisms (SNPs) and amino acid substitutions at the SOD-1 locus, we investigated the functional effects of this variation on SOD-1 activity during ozone stress and subsequent levels of oxidative damage to DNA and cell membranes. Fish from each line were subjected to either control conditions or 24h of ozone stress, after which tissues were analyzed for antioxidant status and oxidative damage. Liver SOD-1 activity was lower in ozonated vs. control fish in the Hot Creek line, and among ozonated fish, Hot Creek was lower than Arlee. Total erythrocyte SOD activity was not significantly impacted by ozonation; however significant differences in total erythrocyte SOD activity were measured among clonal lines, driven primarily by lower activity in the Hot Creek line. Ozone had a significant treatment effect in all oxidative damage parameters assessed: it increased DNA lesions in erythrocytes and levels of lipid peroxidation in gill tissue and plasma. Among lines, Swanson showed higher lipid peroxidation levels in gill tissue after ozonation than Arlee or Hot Creek. Conversely, Swanson control and treatment fish had significantly lower plasma lipid peroxidation levels than did fish from the other lines. Overall, the among-line differences in SOD and SOD-1 activity and oxidative damage provide evidence that SOD-1 genotypes differ functionally under both oxidative stress and control conditions; however, other genetic differences among lines should be investigated in order to further explain the phenotypic differences in SOD enzyme activity and oxidative damage described here.

Sequence divergence was evaluated in the non-recombining, male-specific OmyY1 region of the Y chromosome among the subspecies of cutthroat trout (Oncorhynchus clarkii) in the western United States. This evaluation identified subspecies-discriminating OmyY1-haplotypes within a ∼1200bp region of the OmyY1 locus and localized the region to the end of the Y chromosome by FISH analysis. OmyY1 sequences were aligned and used to reconstruct a phylogeny of the cutthroat trout subspecies and related species via maximum-parsimony and Bayesian analyses. In the Y-haplotype phylogeny, clade distributions generally corresponded to the geographic distributions of the recognized subspecies. This phylogeny generally corresponded to a mitochondrial tree obtained for these subspecies in a previous study. Both support a clade of trout vs. Pacific salmon, of rainbow trout, and of a Yellowstone cutthroat group within the cutthroat trout. In our OmyY1 tree, however, the cutthroat "clade", although present topologically, was not statistically significant. Some key differences were found between trees obtained from the paternally-inherited OmyY1 vs. maternally-inherited mitochondrial haplotypes in cutthroat trout compared to rainbow trout. Other findings are: The trout OmyY1 region evolves between 3 and 13 times slower than the trout mitochondrial regions that have been studied. The Lahontan cutthroat trout had a fixed OmyY1 sequence throughout ten separate populations, suggesting this subspecies underwent a severe population bottleneck prior to its current dispersal throughout the Great Basin during the pluvial phase of the last ice age. The Yellowstone group is the most derived among the cutthroat trout and consists of the Yellowstone, Bonneville, Colorado, Rio Grande and greenback subspecies. Identification of subspecies and sex with this Y-chromosome marker may prove useful in conservation efforts.

In this study, clonal lines from North American resident and migratory populations of rainbow trout Oncorhynchus mykiss adapted to different geographical conditions and with different domestication histories were characterized morphologically. Lines reared in a common-garden experiment were characterized for external shape and meristic values, searching for a general pattern of morphological variation due to exposure to captive conditions. A sharp distinction was identified between wild and captive lines. The body profile was deeper in captive lines, with longer dorsal and anal fins and shorter and deeper caudal peduncles. Highly significant differences were also identified in meristic values among the lines but no consistent relation between meristic values and domestication status was detected. This morphological characterization will facilitate the selection of lines with divergent phenotypes for subsequent quantitative trait loci analysis, aimed at identifying genome regions linked with morphological adaptive response to captive conditions.

Little is known about the factors that drive the high levels of between-host variation in pathogen burden that are frequently observed in viral infections. Here, two factors thought to impact viral load variability, host genetic diversity and stochastic processes linked with viral entry into the host, were examined. This work was conducted with the aquatic vertebrate virus, Infectious hematopoietic necrosis virus (IHNV), in its natural host, rainbow trout. It was found that in controlled in vivo infections of IHNV, a suggestive trend of reduced between-fish viral load variation was observed in a clonal population of isogenic trout compared to a genetically diverse population of out-bred trout. However, this trend was not statistically significant for any of the four viral genotypes examined, and high levels of fish-to-fish variation persisted even in the isogenic trout population. A decrease in fish-to-fish viral load variation was also observed in virus injection challenges that bypassed the host entry step, compared to fish exposed to the virus through the natural water-borne immersion route of infection. This trend was significant for three of the four virus genotypes examined and suggests host entry may play a role in viral load variability. However, high levels of viral load variation also remained in the injection challenges. Together, these results indicate that although host genetic diversity and viral entry may play some role in between-fish viral load variation, they are not major factors. Other biological and non-biological parameters that may influence viral load variation are discussed.

Located in the Pacific Northwest, the Columbia River basin provides important spawning and rearing habitat for Pacific salmon and steelhead (Oncorhynchus spp.). These species were historically abundant throughout the basin but have experienced extensive declines linked to a complex suite of factors. These declines, in tandem with their cultural and economic significance, have led Pacific salmon and steelhead to become one of the most intensely managed groups of species in North America. Management actions have increasingly recognized the importance of genetic resources and have expanded the use of genetic tools to provide powerful data for the conservation and management of Pacific salmon. We provide a summary of historic management actions in the basin with a focus on those relevant to genetic applications. We describe the initial recognition of genetic differences and distinction of population units, how genetics applies to the hatchery controversy, as well as the progression of genetic investigations and applications used in management. Further, we outline some emerging and potential future genetic tools.

The genetic variation of many species in the Northern Hemisphere has been influenced by climatic changes during the Pleistocene Epoch. Phylogeographic studies can help determine intraspecific relationships and postglacial recolonization routes for many of these species, potentially leading to a more complete understanding of how flora and fauna respond to dramatic climate change. We analyzed the variation in the mitochondrial DNA sequences of Chinook salmon Oncorhynchus tshawytscha from California to the Kamchatka Peninsula, Russia, to gain insight into the role of Pleistocene glaciation in the genetic structuring of this species. The geographic distribution of haplotypes revealed high levels of genetic diversity in the Columbia River drainage. Nested clade analysis suggests northward expansion from this area during the late Pleistocene, a hypothesis that is further supported by evidence of demographic growth and population expansion in the northern portion of the species' range. Estimates of the...

In rainbow trout (Oncorhynchus mykiss) and other fishes, embryonic development rate is an ecologically and evolutionarily important trait that is closely associated with survival and physiological performance later in life. To identify genes differentially regulated in fast and slow-developing embryos of rainbow trout, we examined gene expression across developmental time points in rainbow trout embryos possessing alleles linked to a major quantitative trait loci (QTL) for fast versus slow embryonic development rate. Whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family, whereby each backcross generation involved the introgression of the fast-developing alleles for a major development rate QTL into a slow-developing clonal line of rainbow trout. Embryos were collected at 15, 19, and 28 days post-fertilization; sex and QTL genotype were determined using molecular markers, and cDNA from 48 embryos were used for microarray analysis. A total of 183 features were identified with significant differences between embryonic development rate genotypes. Genes associated with cell cycle growth, muscle contraction and protein synthesis were expressed significantly higher in embryos with the fast-developing allele (Clearwater) than those with the slow-developing allele (Oregon State University), which may associate with fast growth and early body mass construction in embryo development. Across time points, individuals with the fast-developing QTL allele appeared to have earlier onset of these developmental processes when compared to individuals with the slow development alleles, even as early as 15 days post-fertilization. Differentially expressed candidate genes chosen for linkage mapping were localized primarily to regions outside of the major embryonic development rate QTL, with the exception of a single gene (very low-density lipoprotein receptor precursor).

Considerable evidence suggests that one genome duplication event preceded the divergence of teleost fishes and a second genome duplication event occurred before the radiation of teleosts of the family Salmonidae. Two Sox9 genes have been isolated from a number of teleosts and are called Sox9a and Sox9b. Two Sox9 gene copies have also been isolated from rainbow trout, a salmonid fish and are called Sox9 and Sox9?2. Previous evaluations of the evolutionary history of rainbow trout Sox9 gene copies using phylogenetic reconstructions of their coding regions indicated that they both belong to the Sox9b clade. In this study, we determine the true evolutionary history of Sox9 gene copies in rainbow trout. We show that the locus referred to as Sox9 in rainbow trout is itself duplicated. Mapping of the duplicated Sox9 gene copies indicates that they are co-orthologs of Sox9b while mapping of Sox9?2 indicates that it is an ortholog of Sox9a. This relationship is supported by phylogenetic reconstruction of Sox9 gene copies in teleosts using their 3? untranslated regions. The conflicting phylogenetic topology of Sox9 genes in rainbow trout indicates the occurrence of gene conversion events between Sox9 and Sox9?2 which is supported by a number of recombination analyses.

Domesticated populations of rainbow trout ( Oncorhynchus mykiss ) probably have been selected for high growth rates and large body size, which may have resulted in reduced sprint swimming performance. We hypothesized that more domesticated populations of rainbow trout would have higher growth rates and larger body size, but slower swim speed, relative to semiwild populations. We tested this hypothesis by measuring body mass, body length, and sprint swim speed multiple times over 92 days in progeny from crosses between males from three clonal lines and an outbred female. We found significantly higher body masses and significantly slower swim speeds in the highly domesticated Arlee and Hot Creek progeny groups compared with the semiwild Swanson hybrid progeny group, supporting our hypothesis. Growth rates also differed significantly among groups, but at the ages measured, the Swanson hybrid progeny had an intermediate growth rate. However, given the differences in body mass, either growth rates were higher in the more domesticated progeny groups at young unmeasured ages and (or) they hatched at a significantly larger body mass than the semiwild Swanson hybrid.

Although cryopreservation is a widely accepted tool in animal breeding and human reproduction, questions have arisen regarding the health and viability of fish sired by cryopreserved sperm. We examined rainbow trout families produced using fresh and cryopreserved milt to determine if sperm cryopreservation negatively influenced early development. Fresh and cryopreserved milt from 3 males were used to fertilize eggs from 6 females in a 3 × 6 factorial design. Survival to eye, survival to fry, proportion of fry deformities and developmental stability were compared for fresh vs. cryopreserved milt. Results revealed a significant reduction in survival to eye ( P P P = 0.127), indicating that survival differences occurred prior to the eye stage. These results could be explained by reduced fertilization success of cryopreserved sperm. We examined developmental differences by analyzing the proportion of haploid embryos at the eyed stage, the proportion of fry deformities at hatch and fluctuating asymmetry (FA) of pectoral fin rays. Results revealed no significant difference between the groups. Our observations that surviving fry produced using cryopreserved sperm showed no differences in early development suggest that sperm cryopreservation is a viable option for use in breeding programs and in conservation and recovery of imperiled salmonid populations.

Bovine chromosome 14 (BTA14) has been widely explored for quantitative trait loci (QTL) and genes related to economically important traits in both dairy and beef cattle. We reviewed more than 40 investigations and anchored 126 QTL to the current genome assembly (Btau 4_0). Using this anchored QTL map, we observed that, in dairy cattle, the region spanning 0 – 10 Mb on BTA14 has the highest density QTL map with a total of 56 QTL, mainly for milk production traits. It is very likely that both somatic cell score (SCS) and clinical mastitis share some common QTL in two regions: 61.48 Mb - 73.84 Mb and 7.86 Mb – 39.55 Mb, respectively. As well, both ovulation rate and twinning rate might share a common QTL region from 34.16 Mb to 65.38 Mb. However, there are no common QTL locations in three pregnancy related phenotypes: non-return rate, pregnancy rate and daughter pregnancy rate. In beef cattle, the majority of QTL are located in a broad region of 15 Mb – 45 Mb on the chromosome. Functional genes, such as CRH, CYP11B1, DGAT1, FABP4 and TG, as potential candidates for some of these QTL, were also reviewed. Therefore, our review provides a standardized QTL map anchored within the current genome assembly, which would enhance the process of selecting positional and physiological candidate genes for many important traits in cattle.

Native inland populations of rainbow trout Oncorhynchus mykiss gairdneri, particularly resident populations, often hybridize with introduced populations of the widely cultured coastal form of the species, O. m. irideus. The inland and coastal subspecies differ genetically from each other by allozyme polymorphisms at the lactate dehydrogenase (LDH-B2*) and superoxide dismutase loci (sSOD-1*) that can be detected using protein electrophoresis. Fewer laboratories, however, are now using allozyme technology, and most genetic studies from wild organisms are now being conducted using DNA rather than protein analyses. We have identified the single-nucleotide polymorphism (SNP) differences responsible for the protein variations by sequencing the complementary DNA for the LDH-B2* and sSOD-1* genes in a large number of individuals whose genotypes were also determined by protein electrophoresis. The genetic differences causing the allozyme polymorphisms have been converted into SNP allelic discrimination as...

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.

Heat shock protein 70 (HSP70, 70 kDa) is the most commonly expressed protein in response to thermal stress. The extent of its expression is associated with differences in environmental temperatures. We investigated the heat shock response in red blood cells collected from one-year-old rainbow trout (Oncorhynchus mykiss). Three different clonal lines of rainbow trout (Arlee, OSU and Whale Rock) were utilized, originating from habitats that likely experienced different thermal profile. The relative expression of HSP70 from blood cells treated at 13 degrees C, 16 degrees C, 18 degrees C, 20 degrees C, 22 degrees C, and 24 degrees C was quantified using real-time PCR. The use of red blood cells allows for the control and replication of HSP70 expression patterns. Relative expression of HSP70 differed significantly among the three clonal lines. The Arlee line had the lowest HSP70 response of the three clonal lines at any temperature; indicating a heritable difference. Maximum expression of HSP70 occurred at 22 degrees C in the OSU line and at 24 degrees C in the Whale Rock line. The discovery of variation in HSP70 expression among the clonal lines indicates that future studies to map the genetic control of HSP70 expression differences are possible.

Major histocompatibility complex (MHC) molecules are important mediators of cell-mediated immunity in vertebrates. MHC class IA molecules are important for host anti-viral immunity as they present intracellular antigens and regulate natural killer cell (NK) activity. MHC class Ib molecules on the other hand are less understood and have demonstrated diverse immune and non-immune functions in mammals. Rainbow trout possess a single classical MHC IA locus (Onmy-UBA) that is believed to function similar to that of mammalian MHC class Ia. Numerous MHC class Ib genes with undetermined functions have also been described in trout. Here we utilize quantitative reverse transcriptase PCR (qRT-PCR) techniques to survey the levels of basal and inducible transcription for selected trout MHC class Ib genes, sIgM and sentinels of IFN induction in response to viral infection. Basal transcription of all the class Ib genes examined in this study was lower than Onmy-UBA in naïve fish. UBA, along with all of the non-classical genes were induced in fish infected with virus but not in control fish. Our results support a non-classical designation for the majority of the class IB genes surveyed in this study based upon expression levels while also indicating that they may play an important role in anti-viral immunity in trout.

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow × cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.

Sex-specific DNA markers are useful for hatchery management. Sex identification at early ages can reduce broodstock rearing costs. This study employed the Amplified Fragment Length Polymorphism (AFLP) approach for the identification of sex-linked markers in Pangasianodon gigas and P. hypophthalmus. Eight DNA pools (4 females, 4 males) each from P. gigas and P. hypophthalmus were screened using a total of 570 and 102 different primer combinations, respectively. None of the 570 primer combinations gave sex-associated amplification for P. gigas while 31 of the 102 primer combinations gave apparent sex-associated amplification across the pooled DNA samples of P. hypophthalmus. However, none of the 45 SCAR markers derived from the presumed sex-specific fragments showed sex specificity when tested using DNA of individual P. hypophthalmus males and females.

Fish species that have external fertilization can be reproduced by induced parthenogenesis. The nuclear content of either the sperm or egg is destroyed by UV or gamma irradiation, and the treated gamete then is fused with an untreated egg or sperm to form a haploid embryo. This is subsequently made diploid by inhibition of either the second meiotic division or the first cell division. After first cell division blockage, the resulting individual is a so-called doubled haploid (DH). DH individuals carry only the duplicated set of chromosomes inherited from the untreated egg or sperm and are, by definition, fully homozygous. In the first part of this review, we discuss the latest insights into the mechanisms underlying the process of making meiotic diploids and DH individuals, and review the problems associated with making and characteristics of doubled haploids and clones in fishes. In the second part of this review, we explore the use of doubled haploids and clones in quantitative trait locus mapping and selective breeding.

Breeding programs may have the unintended consequence of elevating levels of inbreeding; mutations unmasked through inbreeding may have deleterious effects on salmonid population viability. We used induced all-paternal inheritance (androgenesis) to assess the incidence of mutations affecting early development in rainbow trout (Oncorhynchus mykiss) and to provide insights into the genetic basis for inbreeding depression. Assays conducted on androgenetic haploid embryos derived from four rainbow trout populations allowed us to test whether a homozygous clonal line produced in our laboratory had been purged of deleterious alleles affecting early embryonic development. Assays showed no significant difference in viability of androgenetic haploids produced from clonal or outbred lines of rainbow trout during early development. These results are not consistent with the dominance hypothesis of inbreeding depression, which attributes inbreeding depression primarily to expression of deleterious recessive alleles. However, significant maternal effects on haploid embryo viability were identified, implying that egg quality may have a larger effect than paternally inherited deleterious recessive alleles on early haploid embryonic development. Androgenetic haploid assays may thus be useful as a stringent measure of egg quality in aquaculture breeding programs.

Genetic variation in sensitivity to stress is a major determinant of production and welfare of aquaculture species. We investigated the genetics of the stress response using rainbow trout ( Oncorhynchus mykiss ) clonal lines with differing levels of domestication. First, we compared levels of cortisol among three clonal lines: two derived from highly domesticated populations (Arlee and OSU) and one from a population recently taken into captivity (Swanson). The Arlee clonal line showed significantly reduced cortisol levels in response to a netting stressor relative to the OSU and Swanson clonal lines. Next, we used interval mapping and composite interval mapping to scan the genome for quantitative trait loci (QTL) influencing post-stress cortisol levels in doubled haploid offspring of an OSU × Arlee hybrid and compared these to QTL for body mass. Two significant QTL with opposing additive effects on cortisol levels were detected, explaining 43% of the phenotypic variation. Two QTL were also detected for juvenile body mass, one of which overlapped with a QTL for cortisol levels on linkage group OA-XXVII, indicating a possible genetic link between these traits. However, the additive effects of these overlapping QTL suggest a positive relationship between cortisol levels and growth rate rather than the expected negative relationship. Our findings suggest complex genetic control of stress physiology and its relationship with growth rate.

Four complete mitochondrial DNA genomes for rainbow trout, Oncorhynchus mykiss , were sequenced and compared to the previously reported complete mitochondrial sequence. Analyses revealed 13 additional bp and 31 insertion/deletion (indels) sites between the four new sequences and the previously published sequence. Twenty-eight indels were single base pairs while the remaining were multiple base pair insertions, two in the ATPase6 gene, one of three bases and one of nine bases, and a two base insert in the control region. Indels in the new genomes were compared to a wide range of salmonid species and corresponded with sequences in closely related species, indicating that errors were likely incorporated into the previously published sequence. Although variable nucleotide positions were observed throughout the newly sequenced genomes, non-synonymous amino acid substitutions were observed in only six of seven ND genes. While there were only 14 amino acid substitutions between genomes, K a / K s ratios ranged between 0 and 1. A single amino acid deletion in the ND1 coding region unique to rainbow trout was also detected. Using complete mitochondrial genomes for all sequenced Salmonidae species, a phylogenetic analysis was performed to establish a salmonid mitochondrial phylogeny revealing support for Salvelinus , rather than Salmo , as the sister taxa to Oncorhynchus .

People with obesity, especially extreme obesity, are at risk for many health problems. However, the responsible genes remain unknown in >95% of severe obesity cases. Our previous genome-wide scan of Wagyu x Limousin F2 cattle crosses with extreme phenotypes revealed a molecular marker significantly associated with intramuscular fat deposition. Characterization of this marker showed that it is orthologous to the human gene KIAA1462 located on HSA10p11.23, where a major quantitative trait locus for morbid obesity has been reported. The newly identified mitochondrial poly(A) polymerase associated domain containing 1 (PAPD1) gene, which is located near this marker, is particularly interesting because the polymerase is required for the polyadenylation and stabilization of mammalian mitochondrial mRNAs. In the present study, both cDNA and genomic DNA sequences were annotated for the bovine PAPD1 gene and ten genetic markers were detected in the promoter and exon 1 region. Among seven markers assayed on ~ 250 Wagyu x Limousin F2 animals, two single nucleotide polymorphisms (SNPs) in the promoter region were significantly associated with intramuscular fat (P

People with obesity, especially extreme obesity, are at risk for many health problems. However, the responsible genes remain unknown in95% of severe obesity cases. Our previous genome-wide scan of Wagyu x Limousin F2 cattle crosses with extreme phenotypes revealed a molecular marker significantly associated with intramuscular fat deposition. Characterization of this marker showed that it is orthologous to the human gene KIAA1462 located on HSA10p11.23, where a major quantitative trait locus for morbid obesity has been reported. The newly identified mitochondrial poly(A) polymerase associated domain containing 1 (PAPD1) gene, which is located near this marker, is particularly interesting because the polymerase is required for the polyadenylation and stabilization of mammalian mitochondrial mRNAs. In the present study, both cDNA and genomic DNA sequences were annotated for the bovine PAPD1 gene and ten genetic markers were detected in the promoter and exon 1 region. Among seven markers assayed on approximately 250 Wagyu x Limousin F2 animals, two single nucleotide polymorphisms (SNPs) in the promoter region were significantly associated with intramuscular fat (P0.05). However, there was a significant interaction (P0.05) between a third SNP, which causes an amino acid change in coding exon 1, and each of these two promoter SNPs on intramuscular fat deposition. In particular, the differences between double heterozygous animals at two polymorphic sites and the slim genotype animals exceeded 2.3 standard deviations for the trait in both cases. Our study provides evidence for a new mechanism--the involvement of compound heterosis in extreme obesity, which warrants further examination.

Fluorescence in situ hybridization (FISH) was used to identify which chromosome pairs are involved in the difference in karyotypes of three clonal lines of Rainbow Trout (Oncorhynchus mykiss): OSU with 2n = 60, Clearwater with 2n = 58, and Swanson with 2n = 58. These lines appear to have karyotypes typical of strains from the regions from which they were derived (California, Idaho, and Alaska, respectively). Bacterial artificial chromosome clones containing mapped markers specific for different acrocentric pairs in the OSU strain were hybridized to chromosomes of hybrid fish. The OSU × Clearwater and OSU × Swanson hybrids have 2n = 59 with a chromosome pair consisting of one metacentric (fusion) chromosome and two acrocentrics. Both strains have the same chromosome fusion involving acrocentric chromosomes 25 and 29 of the OSU strain although they are from widely separate geographic regions. These results suggest that all of the 2n = 58 Rainbow Trout may have the same karyotype and a common origin.

A DNA AFLP-fingerprinting approach has been used in this study for the identification of sex-linked markers in rainbow trout. Using a sex-typed pool strategy we analyzed a total of 486 different primer combinations of which 19 gave sex-associated amplification across the pooled DNA samples. Segregation analysis showed that 18 out of 19 AFLPs (95%) were informative for individual sexing and genetic mapping. Fifteen AFLPs that showed sex-associated amplification in the pooled segregant analysis were converted into single locus markers (SCARs). One out of 15, the SCAR Omy -163, was Y chromosome-linked and mapped near the sex locus in all of four genetically diverse male clonal lines of rainbow trout examined. Three microsatellites, Ots517NWFSC, OmyFGT19TUF and Omm1026, which were previously mapped on the sex linkage group in rainbow trout, were also analyzed. The SCAR Omy -163, Ots517NWFC and OmyFGT19TUF showed low levels of intraspecific allelic polymorphism. Y chromosome-linked AFLP markers were relatively strain-specific suggesting an intraspecific genetic polymorphism of the rainbow trout Y-chromosome. Estimation of the level of polymorphism for AFLPs using one of our strains revealed that approximately 34,506 putative loci were tested in our study of which about 4374 were polymorphic. Considering 11 Y-linked polymorphic loci in this strain, we estimated that 0.25% of polymorphic loci (11/4374) were confined to a single sex. Assuming that the length of the rainbow trout genome is estimated to be 2627.5 cM (2.4×10 6 kb), the male-determining region detected using our screen may span 6.6 cM (6100 kb). The AFLP technique provides an efficient marker system for revealing polymorphic loci and for linkage map construction. An AFLP approach in combination with a sex-type pool strategy resulted in a quick analysis for identifying sex-linked loci in rainbow trout. Similar approaches may be useful for molecular mapping of markers linked to genes of interest in other species.

The pyloric caeca of fishes are a major site for nutrient digestion and absorption. Some studies have reported an association between increased caeca number, improved feed efficiency and faster overall growth in rainbow trout. However, little is known concerning the genetic basis underlying pyloric caeca development in any fish species. We have identified two clonal rainbow trout lines that differ in pyloric caeca number. Doubled haploid progeny produced from an F 1 hybrid of these lines were used to evaluate the possibility of joint segregation of pyloric caeca phenotype with 330 amplified fragment length polymorphic (AFLP) markers and 39 microsatellites genotyped in 54 individuals. A genetic map has been constructed based on these genotypes and composite interval mapping revealed three major quantitative trait loci (QTL) associated with pyloric caeca number. These QTL respectively explain 19.2%, 18.6% and 13.5% of the variation in pyloric caeca number observed within the doubled haploid progeny. Because these QTL span 3 separate linkage groups, our results demonstrate the phenotype of pyloric caeca number is a polygenic trait in rainbow trout.

Backcrosses of clonal rainbow trout, Oncorhynchus mykiss , were used in an effort for fine-mapping of a quantitative trait locus (QTL) for rapid embryonic development rate ( tth -1). A previously identified QTL was backcrossed for two successive generations from the Clearwater (fast developing) line into the Oregon State University (OSU) (slow developing) background. Microsatellites tightly linked to the QTL and each other were used to genotype individuals in both backcrosses. An association was found between the microsatellites and gamete production in the first backcross and precocious gamete production in the second backcross generation. These results suggest that a region near the QTL-linked microsatellites may influence early sexual maturity, as well as development rate. Two gynogenetic families were produced using individuals from the first backcross (BC 1 ). These crosses were screened with the tth -1 linked microsatellites and time to hatch was quantified. The Clearwater genotype was associated with early time to hatch, while the OSU genotype was associated with late hatching. Genotypic variation at these microsatellite loci explained 28% and 26% of the variation in time to hatch. Heterozygote frequencies for the microsatellites in the two gynogenetic families were 0.0088 and 0.01, suggesting that the QTL is near the centromere. Fine-mapping was limited due to the low level of recombination observed on this linkage group in the gynogenetic families.

Seventeen populations of Oncorhynchus mykiss were analyzed for mitochondrial haplotype diversity through either polymerase chain reaction–restriction fragment length polymorphism analysis or mitochondrial DNA sequencing. Six new mitochondrial haplotypes were observed among these populations, five in steelhead and one in Harney Basin rainbow trout. Although no distinct patterns were observed between steelhead and rainbow trout with mitochondrial DNA sequencing, Harney Basin rainbow trout populations from southeastern Oregon exhibited a high frequency of a unique new haplotype designated MYS22. Fourteen of 17 individuals within the Harney Basin rainbow trout populations possessed this haplotype. Additionally, the mitochondrial DNA haplotypes of six westslope cutthroat trout O. clarki lewisi were observed among 90 steelhead from the Tucannon River, Washington, a river in which no native or introduced cutthroat trout have been documented. Analysis of other introduced hatchery and wild stocks of steel...