To better understand how gene transcription is controlled under different physiological and environmental conditions, we assessed transcription and methylation responses in a vertebrate system (salmon) where growth can be manipulated in response to growth hormone transgenesis or under satiated, feed-deprived, and re-fed experimental conditions. In both transgenic and non-transgenic liver tissue, methylation of gene promoters was negatively associated with transcription at all measured times. However, the changes in promoter methylation among time-points did not generally correlate with changes in gene transcription among treatments. The results from this study suggest that only a subset of genes are readily responsive to changes in promoter methylation for a given environmental shift such as feed-deprivation or a physiological shift such as growth hormone transgenesis (i.e., genotype-by-environment interactions). The present study also reveals the complexity of how one tissue responds to alterations in internal changes in physiology (growth modification by growth hormone transgenesis; genotype), external environmental conditions (food availability; environment), and their interactions (genotype-by-environment effects).

Masu salmon (Oncorhynchus masou) are the only Pacific salmon endemic to Asia. Some researchers prefer to categorize these salmon into 4 subspecies (masu-Oncorhynchus masou masou, amago-Oncorhynchus masou ishikawae, Biwa-Oncorhynchus masou subsp., and Formosan-Oncorhynchus masou formosanus), while others prefer individual species designations. Even though the masu salmon fishery is thousands of years old, classification of the diversity within the masu salmon species complex remains elusive. In this study, a genetic map and reference genome assembly were generated for 1 species/subspecies (masu) to provide resources for understanding the species complex. In O. m. masou, the sex chromosome was determined to be chromosome 7. Resequenced genomes from 2 other putative subspecies (amago and Biwa) provided evidence that they do not share the same sex chromosome. Principal component and admixture analyses clustered the amago and Biwa salmon close together. This supported previous findings of a close relationship between amago and Biwa salmon and a more distant relationship to masu salmon for both. Additional analyses of the masu salmon species complex will benefit from using the new reference genome assembly.

Biocontainment methods for aquaculture are in development and implementation to prevent farmed fish from breeding and thus invading the surrounding environment following an escape from open netpens. Current methods have not demonstrated 100% efficacy in achieving the desired sterility, highlighting the need for the development of novel biocontainment strategies to implement in aquaculture. Using zebrafish as a model organism, we determined the efficacy of the Cre-LoxP system to conditionally express the active form of caspase-3, an apoptosis-inducing cell-death protein, in the developing ovary. This was achieved through the use of two transgenic lines, Tg(zpc:cre; cmlc2-mCherry) and Tg(ef1a:loxP:caspase3:loxP; cmlc2-EGFP), that induce the expression of active caspases in the developing ovary of progeny containing both constructs. Fish positive for only one of the two constructs displayed wildtype (WT) gonadal tissue and bred successfully. However, 78.3% of progeny that carried both constructs did not breed successfully and possessed either little or no gonadal tissue compared to WT controls upon dissection. When combined with induced triploidy, fish that were transgenic for both constructs were 100%. Our results suggest that conditional expression of a cell-death-inducing protein could be used as part of a strategy for the confinement of fish species and demonstrates the utility of "stacking" containment approaches to enhance reproductive containment.

The Fraser River once supported massive salmon returns. However, over the last century, the largest returns have consistently been less than half of the recorded historical maximum. There is substantial interest from surrounding communities and governments to increase salmon returns for both human use and functional ecosystems. To generate resources for this endeavor, we resequenced genomes of Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch), and sockeye salmon (Oncorhynchus nerka) from the Fraser River at moderate coverage (∼16×). A total of 954 resequenced genomes were analyzed, with 681 collected specifically for this study from tissues sampled between 1997 and 2021. An additional 273 were collected from previous studies. At the species level, Chinook salmon appeared to have 1.6-2.1× more SNPs than coho or sockeye salmon, respectively. This difference may be attributable to large historical declines of coho and sockeye salmon. At the population level, 3 Fraser River genetic groups were identified for each species using principal component and admixture analyses. These were consistent with previous research and supports the continued use of these groups in conservation and management efforts. Environmental factors and a migration barrier were identified as major factors influencing the boundaries of these genetic groups. Additionally, 20 potentially adaptive loci were identified among the genetic groups. This information may be valuable in new management and conservation efforts. Furthermore, the resequenced genomes are an important resource for contemporary genomics research on Fraser River salmon and have been made publicly available.

Chum salmon are ecologically important to Pacific Ocean ecosystems and commercially important to fisheries. To improve the genetic resources available for this species, we sequenced and assembled the genome of a male chum salmon using Oxford Nanopore read technology and the Flye genome assembly software (contig N50: ∼2 Mbp, complete BUSCOs: ∼98.1%). We also resequenced the genomes of 59 chum salmon from hatchery sources to better characterize the genome assembly and the diversity of nucleotide variants impacting phenotype variation. With genomic sequences from a doubled haploid individual, we were able to identify regions of the genome assembly that have been collapsed due to high sequence similarity between homeologous (duplicated) chromosomes. The homeologous chromosomes are relics of an ancient salmonid-specific genome duplication. These regions were enriched with genes whose functions are related to the immune system and responses to toxins. From analyzing nucleotide variant annotations of the resequenced genomes, we were also able to identify genes that have increased levels of variants thought to moderately impact gene function. Genes related to the immune system and the detection of chemical stimuli (olfaction) had increased levels of these variants based on a gene ontology enrichment analysis. The tandem organization of many of the enriched genes raises the question of why they have this organization.

In 1969, Professor Toki-o Yamamoto published a landmark review on sex differentiation in fishes, which provided the first comprehensive synthesis of the various influences on sexual phenotype, clarified the nomenclature, and sought to define the mechanisms that determine sex. Yamamoto's review highlighted the genetic basis of sex determination and the influence of sex steroids on gonadal sex differentiation. He argued that genetic sex determination was influenced by the net effect of sex chromosomes, when present, and contributions of female- and male-influencing alleles. Yamamoto also demonstrated that sex steroid treatments could override underlying genetic controls, resulting in sex reversal (e.g., XX-genotype males or XY-genotype females) in gonochoristic species. His comprehensive review has since stimulated a wide range of fields including aquaculture and fisheries sciences, genetics, and aquatic toxicology. This retrospective discusses Yamamoto's scientific legacy and remarkable life, which was devoted to studying the reproductive biology of fishes.

Growth hormone transgenic coho salmon experience increased growth rates, driven primarily through elevated feed intake and feed conversion. However, neuropeptides that signal appetite stimulation have been shown to exhibit variable responses across fed states, suggesting a more complex system mediating growth in these fish. Studies have proposed that growth hormone may have a modulatory role on the energy reserves of fish, possibly through AMP-activated protein kinase (AMPK) activation. AMPK, an energy sensor in cells, has previously been shown to be upregulated in growth hormone transgenic salmon when compared to wild type, however, whether this effect is seen across fed states is unknown. Here, we tested the hypothesis that growth hormone induces an energetic deficit in metabolic tissues, leading to constitutive AMPK activation in growth hormone transgenic salmon. This study compared AMPK activity, ATP, and glycogen, of the liver, heart, and muscle of wild-type, and growth hormone transgenic salmon either fed to satiation or a wild-type ration. The results suggest that white muscle ATP levels in growth hormone salmon are elevated in satiation and rationed conditions. In the liver, growth hormone transgenic salmon fed a rationed wild-type diet experience reductions in ATP level and glycogen. In none of the tissues examined, did AMPK activity change. Taken together, these results indicate that growth hormone transgenic salmon experience metabolic duress when not fed to satiation.

Coho salmon (Oncorhynchus kisutch) are a culturally and economically important species that return from multiyear ocean migrations to spawn in rivers that flow to the Northern Pacific Ocean. Southern stocks of coho salmon in Canada and the United States have significantly declined over the past quarter century, and unfortunately, conservation efforts have not reversed this trend. To assist in stock management and conservation efforts, we generated a chromosome-level genome assembly. We also resequenced the genomes of 83 coho salmon across the North American range to identify nucleotide variants and understand the demographic histories of these salmon by modeling effective population size from genome-wide data. From demographic history modeling, we observed reductions in effective population sizes between 3,750 and 8,000 years ago for several northern sampling sites, which may correspond to bottleneck events during recolonization after glacial retreat.

Doubled haploid gynogens are individuals whose genetic material consists of two identical maternal chromosome sets and who lack paternal genetic contributions. These individuals can be useful in whole-genome sequence assembly to eliminate allelic variation in an individual that otherwise complicates the discrimination of SNPs and paralogs in regular diploids. This is particularly important in salmonid species, which have extensive remnants of an ancestral whole-genome duplication. Further, doubled haploid individuals are fully homozygous and can be used to generate clonal lines. Here, successful timing was determined for late pressure shocking for producing doubled haploid gynogens in five Oncorhynchus species (Chinook Salmon O. tshawytscha, Coho Salmon O. kisutch, Chum Salmon O. keta, Pink Salmon O. gorbuscha, and Sockeye Salmon O. nerka) and one Salvelinus species (domesticated Artic Char Salvelinus alpinus). For this study, sperm was treated with UV irradiation to inactivate the paternal nuclear DNA and used to fertilize eggs. The resulting zygotes were pressure shocked at various times following fertilization to form doubled haploid embryos via inhibition of the first cell division (mitotic gynogenesis). At an incubation temperature of 10.5 degrees C, successful postfertilization pressure shock times for maximal survival of confirmed gynogens were 2.41-2.83 accumulated thermal units (ATUs) for Coho Salmon, 2.63-2.84 ATUs for Chum Salmon, 2.84-3.06 ATUs for Pink Salmon, 2.19 ATUs for Sockeye Salmon, and 2.63-3.06 ATUs for Chinook Salmon, whereas for Arctic Char a shock time of 2.29-2.54 ATUs at 4 degrees C incubation was successful. Survival to fry stage ranged from less than 1% to 11.7%. Survivors were genotyped using microsatellite markers to assess nonpaternity and maternal homozygosity and were found to be 92.5% gynogens when averaged across all treatments and species. Mitotic gynogenetic individuals were produced in all six salmonid species and were used in subsequent studies to generate whole-genome sequences for Chinook, Coho, Chum, and Sockeye salmons.

In fish otoliths, CaCO3 normally precipitates as aragonite, and more rarely as vaterite or calcite. A higher incidence of vaterite deposition in otoliths from aquaculture-reared fish has been reported and it is thought that high growth rates under farming conditions might promote its deposition. To test this hypothesis, otoliths from growth hormone (GH) transgenic coho salmon and non-transgenic fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that transgenic fish otoliths were smaller (-24%, -19%, -20% and -30% for length, width, perimeter and area, respectively; P<0.001) and rounder (-12%, +13.5%, +15% and -15.5% in circularity, form factor, roundness and ellipticity; P<0.001) than otoliths from non-transgenic fish of matching size. Interestingly, transgenic fish had smaller eyes (-30% eye diameter) and showed a strong correlation between eye and otolith size. We also found that the percentage of otoliths showing vaterite deposition was significantly smaller in transgenic fish (21-28%) than in non-transgenic fish (69%; P<0.001). Likewise, the area affected by vaterite deposition within individual otoliths was reduced in transgenic fish (21-26%) compared with non-transgenic fish (42.5%; P<0.001). Our results suggest that high growth rates per se are not sufficient to cause vaterite deposition in all cases, and that GH overexpression might have a protective role against vaterite deposition, a hypothesis that needs further investigation.

Experiments examining potential impacts of growth hormone (GH) transgenesis in fish typically use a single source strain and do not address potential differential impacts in strains of different genetic backgrounds. Here, we examine the effects of differing genetic backgrounds when reared in culture on the growth of transgenic and nontransgenic coho salmon (Oncorhynchus kisutch) produced by mating sires from different rivers with transgenic dams from a single origin. We found a significant difference in size between offspring of sires originating from various river systems in British Columbia. This difference was independent of differences between transgenotypes (i.e., transgenic vs. nontransgenic offspring). However, the effects of strain or sire were relatively small compared to the effects of the transgene, which were consistent regardless of sire origin. Thus, results derived from studies of GH transgenic fish from a single source population can provide useful information for assessments of GH transgenic salmon from other systems. This has important implications for examining potential risks from introgression of a transgene into different populations.

Coho salmon ( Oncorhynchus kisutch ) are a culturally and economically important species that return from multiyear ocean migrations to spawn in rivers that flow to the Northern Pacific Ocean. Southern stocks of coho salmon have significantly declined over the past quarter century, and unfortunately, conservation efforts have not reversed this trend. To assist in stock management and conservation efforts, we generated two chromosome-level genome assemblies and sequenced 24 RNA-seq libraries to better annotate the coho salmon genome assemblies. We also resequenced the genomes of 83 coho salmon across their North American range to identify nucleotide variants, characterize the broad effects of isolation-by-distance using a genome-wide association analysis approach, and understand the demographic histories of these salmon by modeling population size from genome-wide data. We observed that more than 13% of all SNPs were associated with latitude (before multiple test correction), likely an affect of isolation-by-distance. From demographic history modeling, we estimated that the SNP latitudinal gradient likely developed as recently as 8,000 years ago. In addition, we identified four genes each harboring multiple SNPs associated with latitude; all of these SNPs were also predicted to modify the function of the gene. Three of these genes have roles in cell junction maintenance and may be involved in osmoregulation. This signifies that ocean salinity may have been a factor influencing coho salmon recolonization after the last glaciation period – generating the current pattern of variation in these three genes.

Salmonids possess straying abilities that allows them to exploit open territory and establish new populations. Atlantic salmon (Salmo salar) are expanding their distribution primarily as first-generation escapes from aquaculture, whereas Pacific salmon (Oncorhynchus spp.) are expanding their range in polar and temperate regions due to both anthropogenic and natural influences. Here we utilize artificial intergeneric reciprocal crosses to assess the ability of seven species of Pacific salmon to hybridize with Atlantic salmon. Most cross types were found to produce low numbers of hatched embryos, but none survived to sexual maturation. Survivors consisted of diploids and triploids containing both Atlantic and Pacific salmon parental genomes. Thus, introgression of DNA between Pacific and Atlantic salmon may occur to form F 1 hybrids, but transmission to subsequent generations is expected to be rare and occur only over evolutionary time scales. Further, the low viability observed for the most part at early stages of development and in both reciprocal crosses indicates that intergeneric crosses in nature between Atlantic and Pacific salmon are expected to have severe fitness consequences for both dams and sires.

Genotype-by-environment (GxE) interactions are non-parallel reaction norms among individuals with different genotypes in response to different environmental conditions. GxE interactions are an extension of phenotypic plasticity and consequently studying such interactions improves our ability to predict effects of different environments on phenotype as well as the fitness of genetically distinct organisms and their capacity to interact with ecosystems. Growth hormone transgenic coho salmon grow much faster than non-transgenics when raised in tank environments, but show little difference in growth when reared in nature-like streams. We used this model system to evaluate potential mechanisms underlying this growth rate GxE interaction, performing RNA-seq to measure gene transcription and whole-genome bisulfite sequencing to measure gene methylation in liver tissue. Gene ontology (GO) term analysis revealed stress as an important biological process potentially influencing growth rate GxE interactions. While few genes with transcription differences also had methylation differences, in promoter or gene regions, many genes were differentially methylated between tank and stream environments. A GO term analysis of differentially methylated genes between tank and stream environments revealed increased methylation in the stream environment of more than 95% of the differentially methylated genes, many with biological processes unrelated to liver function. The lower nutritional condition of the stream environment may cause increased negative regulation of genes less vital for liver tissue function than when fish are reared in tanks with unlimited food availability. These data show a large effect of rearing environment both on gene expression and methylation, but it is less clear that the detected epigenetic marks are responsible for the observed altered growth and physiological responses.

Chum salmon (Oncorhynchus keta) is the species with the widest geographic range of the anadromous Pacific salmonids,. Chum salmon is the second largest of the Pacific salmon, behind Chinook salmon, and considered the most plentiful Pacific salmon by overall biomass. This species is of significant commercial and economic importance: on average the commercial chum salmon fishery has the second highest processed value of the Pacific salmon within British Columbia. The aim of this work was to establish genomic baseline resources for this species. Our first step to accomplish this goal was to generate a chum salmon reference genome assembly from a doubled-haploid chum salmon. Gene annotation of this genome was facilitated by an extensive RNA-seq database we were able to create from multiple tissues. Range-wide resequencing of chum salmon genomes allowed us to categorize genome-wide geographic variation, which in turn reinforced the idea that genetic differentiation was best described on a regional, rather than at a stock-specific, level. Within British Columbia, chum salmon regional groupings were described at the conservation unit (CU) level, and there may be substructure within particular CUs. Genome wide associations of phenotypic sex to SNP genetic markers identified two clear peaks, a very strong peak on Linkage Group 15, and another on Linkage Group 3. With these new resources, we were better able to characterize the sex-determining region and gain further insights into sex determination in chum salmon and the general biology of this species.

Atlantic and Pacific salmon are frequently consumed species with very different economic values: farmed Atlantic salmon is cheaper than wild-caught Pacific salmons. Species replacements occur with the high valued Pacific species (Oncorhynchus keta, O. gorbuscha, O. kisutch, O. nerka and O. tshawytscha) substituted by cheaper farmed Atlantic salmon (Salmo salar) and Atlantic salmon by rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta). Here we use High-Resolution Melting Analysis (HRMA) to identify eight salmonid species. We designed primers to generate short amplicons of 72 and 116 bp from the fish barcode genes CO1 and CYTB. The time of analysis was under 70 min, after DNA extraction. Food processing of Atlantic salmon (fresh, "Bellevue", "gravadlax", frozen and smoked) did not impact the HRMA profiles allowing reliable identification. A blind test was conducted by three different institutes, showing correct species identifications irrespective of the laboratory conducting the analysis. Finally, a total of 82 retail samples from three European countries were analyzed and a low substitution rate of 1.2% was found. The developed tool provides a quick way to investigate salmon fraud and contributes to safeguard consumers.

Many natural and managed organisms will require substantial functional genetic variation to respond to selection in the face of rapid environmental change. Pacific salmon have experienced strong fluctuations in critical fitness traits over the past five decades. We examined genetic and phenotypic variability over three generations in a pedigreed hatchery population of coho salmon (Oncorhynchus kisutch) by monitoring seven fitness-related traits. Three-year-old adult return numbers varied more than fivefold, and jack (2-year-old males) numbers varied 13-fold. Body sizes of Inch Creek coho salmon decreased consistently such that fish were only 40.7% as heavy in 2015 as in 2006, and female reproductive traits also decreased. During the study period, the majority of families produced returning adult progeny, and effective population size was relatively constant. Heritability estimates for phenotypic traits were significantly greater than zero except for condition factor, and the estimated heritability for jacking was 0.42. The Inch Creek coho salmon population harbours substantial heritability for fitness and reproductive traits and thus likely retains substantial capacity for adaptation despite many years of hatchery propagation.

Growth hormone (GH) transgenic fish often exhibit remarkable transformations in growth rate and other phenotypes relative to wild-type. The 5750A transgenic coho salmon strain exhibits strong sexually dimorphic growth, with females possessing growth stimulation at a level typical of that seen for both sexes in other strains harbouring the same gene construct (e.g. M77), while males display a modest level of growth stimulation. GH mRNA levels were significantly higher in females than in males of the 5750A strain but equivalent in the M77 strain, indicating sex and transgene insertion locus altered transgene expression. We found that acute estradiol treatments did not influence GH expression in either strain (5750A and M77) or the transgene promoter (metallothionein-B), suggesting that estradiol level was not a significant factor influencing transgene activity. The feminization of XX and XY fish of the 5750A and M77 strains generated all-female groups and resulted in equalized growth of the two genetic sexes, suggesting that the presence of the Y chromosome was not directly capable of influencing the GH transgene–mediated growth in a physiological female conditions. These data suggest that the difference in growth rate seen between the sexes in the 5750A strain arises from non-estradiol-mediated sex influences on gene regulation at the transgene locus. This study shows how genetic factors and transgene insertion sites can influence transgene expression with significant consequent effects on phenotype.