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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.

Background Growth regulation is a complex process influenced by genetic and environmental factors. We examined differences between growth hormone (GH) transgenic (T) and non-transgenic (NT) coho salmon to elucidate whether the same loci were involved in controlling body size and gene expression phenotypes, and to assess whether physiological transformations occurring from GH transgenesis were under the influence of alternative pathways. The following genomic techniques were used to explore differences between size classes within and between transgenotypes (T vs. NT): RNA-Seq/Differentially Expressed Gene (DEG) analysis, quantitative PCR (qPCR) and OpenArray analysis, Genotyping-by-Sequencing, and Genome-Wide Association Study (GWAS). Results DEGs identified in comparisons between the large and small tails of the size distributions of T and NT salmon (NTLarge, NTSmall, TLarge and TSmall) spanned a broad range of biological processes, indicating wide-spread influence of the transgene on gene expression. Overexpression of growth hormone led to differences in regulatory loci between transgenotypes and size classes. Expression levels were significantly greater in T fish at 16 of 31 loci and in NT fish for 10 loci. Eleven genes exhibited different mRNA levels when the interaction of size and transgenotype was considered (IGF1, IGFBP1, GH, C3–4, FAS, FAD6, GLUT1, G6PASE1, GOGAT, MID1IP1). In the GWAS, 649 unique SNPs were significantly associated with at least one study trait, with most SNPs associated with one of the following traits: C3_4, ELA1, GLK, IGF1, IGFBP1, IGFII, or LEPTIN. Only 1 phenotype-associated SNP was found in common between T and NT fish, and there were no SNPs in common between transgenotypes when size was considered. Conclusions Multiple regulatory loci affecting gene expression were shared between fast-growing and slow-growing fish within T or NT groups, but no such regulatory loci were found to be shared between NT and T groups. These data reveal how GH overexpression affects the regulatory responses of the genome resulting in differences in growth, physiological pathways, and gene expression in T fish compared with the wild type. Understanding the complexity of regulatory gene interactions to generate phenotypes has importance in multiple fields ranging from applications in selective breeding to quantifying influences on evolutionary processes.

Background Transcriptomic responses to immune stimulation were investigated in coho salmon (Oncorhynchus kisutch) with distinct growth phenotypes. Wild-type fish were contrasted to strains with accelerated growth arising either from selective breeding (i.e. domestication) or genetic modification. Such distinct routes to accelerated growth may have unique implications for relationships and/or trade-offs between growth and immune function. Results RNA-Seq was performed on liver and head kidney in four ‘growth response groups’ injected with polyinosinic-polycytidylic acid (Poly I:C; viral mimic), peptidoglycan (PGN; bacterial mimic) or PBS (control). These groups were: 1) ‘W’: wild-type, 2) ‘TF’: growth hormone (GH) transgenic salmon with ~ 3-fold higher growth-rate than W, 3) ‘TR’: GH transgenic fish ration restricted to possess a growth-rate equal to W, and 4) ‘D’: domesticated non-transgenic fish showing growth-rate intermediate to W and TF. D and TF showed a higher similarity in transcriptomic response compared to W and TR. Several immune genes showed constitutive expression differences among growth response groups, including perforin 1 and C-C motif chemokine 19-like. Among the affected immune pathways, most were up-regulated by Poly I:C and PGN. In response to PGN, the c-type lectin receptor signalling pathway responded uniquely in TF and TR. In response to stimulation with both immune mimics, TR responded more strongly than other groups. Further, group-specific pathway responses to PGN stimulation included NOD-like receptor signalling in W and platelet activation in TR. TF consistently showed the most attenuated immune response relative to W, and more DEGs were apparent in TR than TF and D relative to W, suggesting that a non-satiating ration coupled with elevated circulating GH levels may cause TR to possess enhanced immune capabilities. Alternatively, TF and D salmon are prevented from acquiring the same level of immune response as TR due to direction of energy to high overall somatic growth. Further study of the effects of ration restriction in growth-modified fishes is warranted. Conclusions These findings improve our understanding of the pleiotropic effects of growth modification on the immunological responses of fish, revealing unique immune pathway responses depending on the mechanism of growth acceleration and nutritional availability.

Background Triploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild. Results The vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state. Conclusions These findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature. Electronic supplementary material The online version of this article (10.1186/s12864-019-5655-8) contains supplementary material, which is available to authorized users.

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.

Pink salmon (Oncorhynchus gorbuscha) adults are the smallest of the five Pacific salmon native to the western Pacific Ocean. Pink salmon are also the most abundant of these species and account for a large proportion of the commercial value of the salmon fishery worldwide. A two-year life history of pink salmon generates temporally isolated populations that spawn either in even-years or odd-years. To uncover the influence of this genetic isolation, reference genome assemblies were generated for each year-class and whole genome re-sequencing data was collected from salmon of both year-classes. The salmon were sampled from six Canadian rivers and one Japanese river. At multiple centromeres we identified peaks of Fst between year-classes that were millions of base-pairs long. The largest Fst peak was also associated with a million base-pair chromosomal polymorphism found in the odd-year genome near a centromere. These Fst peaks may be the result of a centromere drive or a combination of reduced recombination and genetic drift, and they could influence speciation. Other regions of the genome influenced by odd-year and even-year temporal isolation and tentatively under selection were mostly associated with genes related to immune function, organ development/maintenance, and behaviour.

Sockeye salmon (Oncorhynchus nerka) is a commercially and culturally important species to the people that live along the northern Pacific Ocean coast. There are two main sockeye salmon ecotypes-the ocean-going (anadromous) ecotype and the fresh-water ecotype known as kokanee. The goal of this study was to better understand the population structure of sockeye salmon and identify possible genomic differences among populations and between the two ecotypes. In pursuit of this goal, we generated the first reference sockeye salmon genome assembly and an RNA-seq transcriptome data set to better annotate features of the assembly. Resequenced whole-genomes of 140 sockeye salmon and kokanee were analyzed to understand population structure and identify genomic differences between ecotypes. Three distinct geographic and genetic groups were identified from analyses of the resequencing data. Nucleotide variants in an immunoglobulin heavy chain variable gene cluster on chromosome 26 were found to differentiate the northwestern group from the southern and upper Columbia River groups. Several candidate genes were found to be associated with the kokanee ecotype. Many of these genes were related to ammonia tolerance or vision. Finally, the sex chromosomes of this species were better characterized, and an alternative sex-determination mechanism was identified in a subset of upper Columbia River kokanee.

Sex-biased predation, a predator’s bias for one prey sex over the other, can have important demographic impacts on prey species of conservation concern. Yet, it is difficult to measure in the wild. Molecular scatology has been used to indirectly determine the proportion of prey items consumed in the diet, and it may be possible to apply similar approaches to determine the sex-biased consumption of prey items. We developed a molecular method to indirectly examine sex-specific predation employing scat, focusing on predator–prey interactions between Chinook salmon (Oncorhynchus tshawytscha) and harbor seals (Phoca vitulina richardii). We established that the proportions of male and female Chinook DNA can be determined in a controlled sample by measuring a Y-linked marker, growth hormone pseudogene, using qPCR. We then applied the assay to harbor seal scat samples. Although the assay amplified in 83% of scat samples, 27% of samples quantified had an estimated male proportion > 1, which may have been due to a lack of robustness of the PCR assay in samples. Lastly, we constructed a biomass calibration curve to determine whether DNA measurements could estimate the proportions of male and female biomass consumed. The calibration curve was skewed by high male DNA density precluding our ability to quantify the relative amounts. We demonstrated that nuclear prey markers can be amplified in predator scat, however, contamination and extreme DNA density differences between the prey sexes may pose practical difficulties to estimate the relative amounts of male to female biomass consumed.

Next generation sequencing data can be used for rapid identification of genetic markers linked to phenotypes of interest for use in conservation, resource management or breeding. Here we report on the use of genotyping by sequencing (GBS) to develop SNP markers linked with phenotypic sex and with a transgenic construct in coho salmon (Oncorhynchus kisutch). Associations between phenotypes and genotypes were confirmed using high-resolution melting analysis (HRMA). A total of 56 SNPs were associated with phenotypic sex after analysis with the software TASSEL. Concordance between phenotypes and genotypes were tested for 28 SNPS, and of these, seven had moderate to high concordance across test populations. However, only one SNP showed 100 % concordance across all test populations. Fifteen SNPs were associated with the transgenotype using a TASSEL analysis of the GBS data; of these, four showed moderate to high concordance with the transgenotype using HRMA. This type of approach could be used to identify SNPs associated with phenotypes of interest in species with few previously developed genetic resources.

Pacific salmonid (Oncorhynchusspp.) remains are routinely recovered from archaeological sites in northwestern North America but typically lack sexually dimorphic features, precluding the sex identification of these remains through morphological approaches. Consequently, little is known about the deep history of the sex-selective salmonid fishing strategies practiced by some of the region’s Indigenous peoples. Here, we present a DNA-based method for the sex identification of archaeological Pacific salmonid remains that integrates two PCR assays that each co-amplify fragments of thesexually dimorphic on the Y chromosome(sdY) gene and an internal positive control (Clock1aor D-loop). The first assay co-amplifies a 95 bp fragment ofsdYand a 108 bp fragment of the autosomalClock1agene, whereas the second assay co-amplifies the samesdYfragment and a 249 bp fragment of the mitochondrial D-loop region. This method’s reliability, sensitivity, and efficiency, were evaluated by applying it to 72 modern Pacific salmonids from five species and 75 archaeological remains from six Pacific salmonids. The sex identities assigned to each of the modern samples were concordant with their known phenotypic sex, highlighting the method’s reliability. Applications of the method to dilutions of modern DNA samples indicate it can correctly identify the sex of samples with as little as ~39 pg of total genomic DNA. The successful sex identification of 70 of the 75 (93%) archaeological samples further demonstrates the method’s sensitivity. The method’s reliance on two co-amplifications that preferentially amplifysdYhelps validate the sex identities assigned to samples and reduce erroneous identifications caused by allelic dropout and contamination. Furthermore, by sequencing the D-loop fragment used as a positive control, species-level and sex identifications can be simultaneously assigned to samples. Overall, our results indicate the DNA-based method reported in this study is a sensitive and reliable sex identification method for ancient salmonid remains.

Most fish species do not have single-chromosome-based sex determination or display cytologically distinguishable sex chromosomes. The selective forces acting on homologous sequences in diploid autosomal versus haploid sex-chromosomal regions are expected to be distinct and thus to differentially influence genetic variation. In Chinook Salmon Oncorhynchus tshawytscha, the Y chromosome possesses a growth hormone pseudogene (ghp) that is linked to the sex-determination locus and is derived from the functional autosomal growth hormone 2 gene (gh2). Thus, examining these two paralogues provides a model with which to study the forces affecting the persistence of genetic variation between sex-linked and autosomal loci among individuals in Chinook Salmon populations. We characterized single-nucleotide polymorphisms in a 1.6-kb contiguous homologous region in gh2 and ghp in 315 individuals from 19 Chinook Salmon populations ranging from Russia to Alaska, British Columbia, and California. The ghp sequence w...

Whole genome duplication has been implicated in evolutionary innovation and rapid diversification. In salmonid fishes, however, whole genome duplication significantly pre-dates major transitions across the family, and re-diploidization has been a gradual process between genomes that have remained essentially collinear. Nevertheless, pairs of duplicated chromosome arms have diverged at different rates from each other, suggesting that the retention of duplicated regions through occasional pairing between homeologous chromosomes may have played an evolutionary role across species pairs. Extensive chromosomal arm rearrangements have been a key mechanism involved in re-dipliodization of the salmonid genome; therefore, we investigated their influence on degree of differentiation between homeologs across salmon species. We derived a linkage map for coho salmon and performed comparative mapping across syntenic arms within the genus Oncorhynchus, and with the genus Salmo, to determine the phylogenetic relationship between chromosome arrangements and the retention of undifferentiated duplicated regions. A 6596.7 cM female coho salmon map, comprising 30 linkage groups with 7415 and 1266 nonduplicated and duplicated loci, respectively, revealed uneven distribution of duplicated loci along and between chromosome arms. These duplicated regions were conserved across syntenic arms across Oncorhynchus species and were identified in metacentric chromosomes likely formed ancestrally to the divergence of Oncorhynchus from Salmo. These findings support previous studies in which observed pairings involved at least one metacentric chromosome. Re-diploidization in salmon may have been prevented or retarded by the formation of metacentric chromosomes after the whole genome duplication event and may explain lineage-specific innovations in salmon species if functional genes are found in these regions.

Growth rate can be genetically modified in many vertebrates by domestication and selection and more recently by transgenesis overexpressing growth factor genes [e.g., growth hormone (GH)]. Although the phenotypic end consequence is similar, it is currently not clear whether the same modifications to physiological pathways are occurring in both genetic processes or to what extent they may interact when combined. To investigate these questions, microarray analysis has been used to assess levels of mRNA in liver of wild-type and growth-modified strains of rainbow trout (Oncorhynchus mykiss). This species has been used as a model because nondomesticated wild strains are available as comparators to assess genetic and physiological changes that have arisen both from domestication and from GH transgenesis. The analysis examined pure wild-type and pure domesticated strains as well as 2 different GH transgenes (with markedly different growth effects) both in pure wild and in wild × domesticated hybrid backgrounds. Liver mRNA showed highly concordant changes (Pearson correlations; r>0.828; P

To enhance understanding of triploid gene expression, the transcriptome information from bighead catfish (Clarias macrocephalus Gunther, 1864) was studied using the paired-end Illumina HiSeq™ 2000 sequencing platform. In total, 68,227,832 raw reads were generated from liver tissues and 53,149 unigenes were assembled, with an average length of 765 bp and N50 length of 1283 bp. Of these unigenes, 33,428 (62.89%) could be annotated according to their homology with matches in the NCBI non-redundant (Nr), NCBI nucleotide (Nt), Swiss-Prot, Clusters of Orthologous Groups (COG), gene ontology (GO), or Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Relative expression of liver genes between diploid and triploid bighead catfish revealed more than 90% of the annotated unigenes similarly expressed, regardless of ploidy, whereas 362 upregulated and 83 downregulated with at least a twofold change in triploid relative to diploid. Quantitative real-time PCR of 15 differentially expressed growth-related genes showed consistency between the expression profiles of those genes with the results from RNA-seq analysis. Our results showed that genes in C. macrocephalus liver responded independently to triploidy with the majority showing similar expression levels between diploid and triploid (a dosage compensation phenomenon). The underlying mechanism of the varying gene expression patterns was discussed. Notably, 5 of the top 20 upregulated genes associated with stress response and thus may reflect stress caused by triploidy. The present study adds a substantial contribution to the sequence data available for C. macrocephalus and hence provides valuable resources for further studies. Furthermore, it gives information that may enhance understanding of triploid physiology.

We tested the fitness consequences of introgression of fast-growing domesticated fish into a wild population. Fry from wild and domesticated rainbow trout (Oncorhynchus mykiss) crosses, F1 hybrids, and first- and second-generation backcrosses were released into two natural lakes. Parentage analysis using microsatellite loci facilitated the identification of survivors, so fitness was estimated in nature from the first-feeding stage. Results indicated that under certain conditions, domesticated fish survived at least as well as wild fish within the same environment. Relative growth and survival of the crosses, however, were highly dependent on environment. During the first summer, fastest-growing crosses had the highest survival, but this trend was reversed after one winter and another summer. Although the F1 hybrids showed evidence of outbreeding depression because of the disruption of local adaptation, there was little evidence of outbreeding depression in the backcrosses, and the second-generation backcrosses exhibited a wild-type phenotype. This information is relevant for assessing the multigenerational risk of escaped or released domesticated fish should they successfully interbreed with wild populations and provides information on how to minimize detrimental impacts of a conservation breeding and/or management programme. These data also further understanding of the selection pressures in nature that maintain submaximal rates of growth.

Rigorous evaluation of the utility of captive breeding for the restoration of depleted wild salmonid fish populations has not been undertaken. In particular, little is known about the reproductive success of captively-bred individuals that are released back into an extant population and their capacity to assist in long-term population persistence. For the endangered Cultus Lake sockeye salmon population, we examined the potential genetic contribution of the first juvenile fish released from a captive breeding program upon their maturity in the natural Cultus Lake environment. Genetic analysis of 792 Cultus sockeye salmon that were spawned in captivity in 2004 and their adult progeny of 2007 and 2008 revealed a genetic bottleneck originating from 20 wild sockeye salmon hatchery-spawned at Cultus Lake in the previous generation. Pedigree analysis revealed that six of the 20 sockeye salmon spawned in 2001 (grandparents) gave rise to a majority of the hatchery spawners in 2004 (parents) and provided more than 30% of the genes in the progeny that survived to maturity in the wild. Allele frequencies and genetic diversity of the age three progeny that returned to Cultus Lake from their marine migration in 2007 reflected the bottleneck, but its genetic signature was faint among the more genetically diverse age four fish that returned in 2008. Two-generation analysis of gene origin among fish resulting from 2004 hatchery production indicated that they contained the genetic diversity expected from 36 effective ancestors.

The production of growth hormone (GH) transgenic animals has raised a host of social and scientific concerns regarding their potential impacts on ecosystems should they escape into nature. Indeed, theoretical models suggest that GH transgenic animals could, under specific conditions, decimate local populations. However, while there are numerous laboratory examinations of factors affecting survival of transgenic and wild animals, we know little about the competitive reproductive capacity of transgenic animals. Here, we examined the reproductive capabilities of cultured GH transgenic coho salmon (Oncorhynchus kisutch) when in competition with wild coho derived from nature using semi-natural mating arenas (within a contained facility). To account for the well-known reproductive impairments associated with culturing salmon in laboratory facilities we contrasted the competitive reproductive success of GH transgenic coho against that of cultured non-transgenic coho. We also performed in vitro sperm analyses to assess the postcopulatory competitive ability of GH transgenic coho. In competitive mating arenas, transgenic coho performed fewer courtship and aggressive behaviours than coho from nature and sired less than 6% of offspring. Non-transgenic cultured coho, despite their smaller body size, sired more than twice as many offspring than transgenic coho when competing against wild coho in mating arenas. Transgenic males also face a postcopulatory reproductive disadvantage as their ejaculates contained fewer sperm that swam slower and for shorter durations than sperm from wild males. Together, these findings suggest limited potential for the transmission of transgenes from cultured GH transgenic coho salmon through natural matings should they escape from a contained culture facility into nature and reproductively interact with a local wild coho salmon strain. However, as responses of wild-reared fish can differ greatly from those of cultured fish, we stress the importance of understanding genotype-by-environment interactions for reproductive phenotypes when developing risk assessment information. (C) 2011 Elsevier B.V. All rights reserved.

Genetically modified strains usually are generated within defined genetic backgrounds to minimize variation for the engineered characteristic in order to facilitate basic research investigations or for commercial application. However, interactions between transgenes and genetic background have been documented in both model and commercial agricultural species, indicating that allelic variation at transgene-modifying loci are not uncommon in genomes. Engineered organisms that have the potential to allow entry of transgenes into natural populations may cause changes to ecosystems via the interaction of their specific phenotypes with ecosystem components and services. A transgene introgressing through natural populations is likely to encounter a range of natural genetic variation (among individuals or sub-populations) that could result in changes in phenotype, concomitant with effects on fitness and ecosystem consequences that differ from that seen in the progenitor transgenic strain. In the present study, using a growth hormone transgenic salmon example, we have modeled selection of modifier loci (single and multiple) in the presence of a transgene and have found that accounting for genetic background can significantly affect the persistence of transgenes in populations, potentially reducing or reversing a “Trojan gene” effect. Influences from altered life history characteristics (e.g., developmental timing, age of maturation) and compensatory demographic/ecosystem controls (e.g., density dependence) also were found to have a strong influence on transgene effects. Further, with the presence of a transgene in a population, genetic backgrounds were found to shift in non-transgenic individuals as well, an effect expected to direct phenotypes away from naturally selected optima. The present model has revealed the importance of understanding effects of selection for background genetics on the evolution of phenotypes in populations harbouring transgenes.

The present study has assessed the efficacy of spontaneous and pressure-shock-induced triploidy among 60,366 transgenic coho salmon progeny derived from crosses between homozygous growth hormone (GH) transgenic fathers and wild-type mothers. Trials found no effect of pressure, treatment timing post-fertilization, treatment duration, or incubation temperature on the presence of diploid exception frequency, whereas older eggs (held in vivo or in vitro ) were found to have lower frequencies of triploid failure. High temperature and young egg age were found to reduce survival of treated eggs, and family effects influenced survival in many treatments. Spontaneous triploidy was found to be rare (0.06% and 0.22% when eggs were stored in vivo or in vitro , respectively). Three larger scale trials ( n = 15,814, 10,419, and 19,593) using normal pressure, high pressure, or high pressure plus aged eggs, yielded triploidy frequencies of 99.8%, 97.6%, and 97.0%, respectively. Overall, among all pressure-treated groups ( n = 54,787 fish), 1.1% exceptional diploids were detected. If families with obvious high levels (>2%) of diploids are excluded, the frequency of diploid exceptions is 0.32%. Analyses of maternal genetic contributions to shocked progeny were consistent with 100% retention of the second polar body both in diploid exceptional individuals and in triploids. However, diploid exceptions did not consistently possess paternal genetic information (for GH transgene and microsatellite loci), and appeared to be either diploids or hyperdiploid aneuploids (i.e. possessing some but not all of the paternal marker loci). Thus, although sperm appear to fertilize and activate egg development in diploid exceptions, paternal chromosomes are not consistently contributed to the developing zygote's genome. It will be important to assess the fertility of these exceptional “diploid” individuals to determine whether rare cytological events occurring at fertilization may influence the utility of pressure-shock-induced triploidy for biocontainment purposes.

When unifying genomic resources among studies and comparing data between species, there is often no better resource than a genome sequence. Having a reference genome for the Chinook salmon (Oncorhynchus tshawytscha) will enable the extensive genomic resources available for Pacific salmon, Atlantic salmon, and rainbow trout to be leveraged when asking questions related to the Chinook salmon. The Chinook salmon's wide distribution, long cultural impact, evolutionary history, substantial hatchery production, and recent wild-population decline make it an important research species. In this study, we sequenced and assembled the genome of a Chilliwack River Hatchery female Chinook salmon (gynogenetic and homozygous at all loci). With a reference genome sequence, new questions can be asked about the nature of this species, and its role in a rapidly changing world.

Very little information is currently available regarding the sites of integration of transgenes in genetically engineered fish. We examined the chromosomal location of growth hormone gene constructs containing GH1 in three different strains of transgenic coho salmon produced by microinjection into pronuclei of fertilized eggs. The constructs were labeled and used as probes in fluorescence in situ hybridization experiments on chromosome preparations from the M77, MT5750A, and H3D0474 strains of transgenic coho salmon. The constructs were localized at 1–3 different sites in different strains. In the M77 strain the construct was found at a single centromeric site on a medium-sized metacentric chromosome, while in the MT5750A strain, the construct was found at a single telomeric site on the short arm of chromosome pair 21, a subtelocentric chromosome with a large band of repetitive DNA on the short arm. In the H3D0474 strain, the construct was found at telomeric sites on the long arms of three metacentric chromosomes that appear to represent one pair of homologous chromosomes and one chromosome containing the homeologous long arm (recently duplicated chromosome arm) corresponding to the long arm of the first pair. This suggests transfer of the construct may have occurred by homologous and homeologous crossing over. All of the constructs incorporated at restricted sites characterized by the presence of tandem DNA repeats.

Domestication has produced faster-growing strains of animals for use in agriculture, but selection has been applied with little knowledge of the underlying genetic changes that arose throughout the process. Mammals and birds have been domesticated for thousands of years whereas fish have been domesticated only recently; therefore, wild progenitor strains remain for comparison. Rainbow trout (Oncorhynchus mykiss) have undergone intensive selection and domesticated strains grow more rapidly than extant wild strains. To assess physiological pathways altered by domestication, whole-genome mRNA expression was measured in brain, muscle and liver of size-matched domestic and wild trout using a 16K (cGRASP) salmonid microarray. A large number of genes differed between strains, ranging from 3% of genes in brain to 9% in muscle. Domestic fish had more down-regulated genes in the brain relative to wild fish, whereas more genes were up-regulated in domestic liver and muscle. Relative to wild fish, there was a down-regulation of cell division and an up-regulation of structural genes in the brain of domestic fish. In liver from domestic fish, there was an up-regulation of genes related to transport with a down-regulation of lipid binding. Analysis of the functional categories for muscle indicated that most pathways, including pathways related to metabolism and catabolism, were up-regulated in domestic fish. Comparison of these results to other genomic studies on transgenic, domestic and wild salmonids suggests that similar physiological pathways are altered systemically to support faster rates of growth, regardless of the underlying genetic alteration that has caused the altered growth.

Carotenoids constitute a vast group of pigments that are ubiquitous throughout nature. Carrot (Daucus carota L.) roots provide an important source of dietary beta-carotene (provitamin A), alpha-carotene and lutein. Ketocarotenoids, such as canthaxanthin and astaxanthin, are produced by some algae and cyanobacteria but are rare in plants. Ketocarotenoids are strong antioxidants that are chemically synthesized and used as dietary supplements and pigments in the aquaculture and neutraceutical industries. We engineered the ketocarotenoid biosynthetic pathway in carrot tissues by introducing a beta-carotene ketolase gene isolated from the alga Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV 35S, Arabidopsis-ubiquitin, and RolD from Agrobacterium rhizogenes). The pea Rubisco small sub-unit transit peptide was used to target the enzyme to plastids in leaf and root tissues. The phosphinothricin acetyl transferase (bar) gene was used as a selectable marker. Following Agrobacterium-mediated transformation, 150 plants were regenerated and grown in a glasshouse. All three promoters provided strong root expression, while the double CaMV 35S and Ubiquitin promoters also had strong leaf expression. The recombinant ketolase protein was successfully targeted to the chloroplasts and chromoplasts. Endogenous expression of carrot beta-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of total carotenoids was converted to novel ketocarotenoids, with accumulation up to 2,400 microg/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin and beta-cryptoxanthin. Our results show that carrots are suitable for biopharming ketocarotenoid production for applications to the functional food, neutraceutical and aquaculture industries.

The purpose of this study was to examine, using a rainbow trout (Oncorhynchus mykiss) model system, the fitness consequences of three generations of introgression of genotypes adapted to two different environments (culture and nature). The experiments also isolated the influence of competitive interactions and risk of predation on the relative growth and survival of the wild and backcrossed lines. Line crosses representing fast-growing pure domestic (D), slow-growing pure wild (W), domestic x wild hybrids (F1), F1 x wild backcrosses (B1), and B1 x wild backcrosses (B2) were generated and reared under (1) culture conditions, (2) seminatural conditions with competition among genotypes, and (3) seminatural conditions under risk of predation. Survival of the fry in a seminatural environment with competition fit an additive model of gene action with the domestic fish having the highest survival and the wild fish the lowest, but under risk of predation outbreeding depression was suggested by low survival of the B2 lines. Evidence of a trade-off in growth and survival under risk of predation along with observations of genetically determined behavioral differences among the strains may provide some explanation for the observed differences in survival among the strains. This information is relevant to improving our evolutionary understanding of the interaction among genomes, and the influence of environment, during hybridization events. Results from this experiment indicate that alteration of phenotype likely played a prominent role in the reduced fitness experienced by progeny produced after three generations of introgression, supporting the theory that disruption of genotypes selected for adaptation to local conditions may be a primary cause of outbreeding depression in species such as salmon.

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in the karyotypes of chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha). The sex chromosome pair is a small acrocentric chromosome pair in chum salmon and the smallest metacentric chromosome pair in pink salmon. Both of these chromosome pairs are morphologically different from the sex chromosome pairs in chinook salmon (Oncorhynchus tshawytscha) and coho salmon (Oncorhynchus kisutch). The 5S rRNA genes are on multiple chromosome pairs including the sex chromosome pair in chum salmon, but at the centromeres of two autosomal metacentric pairs in pink salmon. The sex chromosome pairs and the chromosomal locations of the 5S rDNA appear to be different in all five of the North American Pacific salmon species and rainbow trout. The implications of these results for evolution of sex chromosomes in salmonids are discussed.

Chinook salmon (Oncorhynchus tshawytscha) exhibit extreme differences in coloration of skin, eggs and flesh due to genetic polymorphisms affecting carotenoid deposition, where colour can range from white to bright red. A sympatric population of red and white Chinook salmon occurs in the Quesnel River, British Columbia, where frequencies of each phenotype are relatively equal. In our study, we examined evolutionary mechanisms responsible for the maintenance of the morphs, where we first tested whether morphs were reproductively isolated using microsatellite genotyping, and second, using breeding trials in seminatural spawning channels, we tested whether colour assortative mate choice could be operating to maintain the polymorphism in nature. Next, given extreme difference in carotenoid assimilation and the importance of carotenoids to immune function, we examined mate choice and selection between colour morphs at immune genes (major histocompatibility complex genes: MHC I-A1 and MHC II-B1). In our study, red and white individuals were found to interbreed, and under seminatural conditions, some degree of colour assortative mate choice (71% of matings) was observed. We found significant genetic differences at both MHC genes between morphs, but no evidence of MHC II-B1-based mate choice. White individuals were more heterozygous at MHC II-B1 compared with red individuals, and morphs showed significant allele frequency differences at MHC I-A1. Although colour assortative mate choice is likely not a primary mechanism maintaining the polymorphisms in the population, our results suggest that selection is operating differentially at immune genes in red and white Chinook salmon, possibly due to differences in carotenoid utilization.

Salmonids are an important family of fish both from economic and basic research perspectives, and have been subjected to extensive research at whole-animal and molecular levels. Most research to date has been conducted on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), but more recently other salmonids have become a focus of study due to their interesting life histories and because of their potential for use in commercial aquaculture. However, molecular biology and genetic analyses for these emerging species are currently hampered due to the lack of extensive genomic resources. To overcome some of these limitations, we have constructed a 43,228 sequence transcriptome from 13 tissues from coho salmon, Oncorhynchus kisutch using de novo transcriptome assembly methods. The transcriptome profiling analysis has provided data distinguishing allelic variation from paralogues that arose during the recent whole-genome duplication event in this family, thus allowing simplified analysis of gene-specific expression. Additionally, 1599 novel coho sequences have been identified through comparison with transcriptomes from two other salmonids species (Atlantic salmon and rainbow trout), and with northern pike. The transcriptome presented here will be useful for genomic analysis of coho salmon and other closely related salmonid species.

Background Individuals rarely grow as fast as their physiologies permit despite the fitness advantages of being large. One reason may be that rapid growth is costly, resulting for example in somatic damage. The chromosomal ends, the telomeres, are particularly vulnerable to such damage, and telomere attrition thus influences the rate of ageing. Here, we used a transgenic salmon model with an artificially increased growth rate to test the hypothesis that rapid growth is traded off against the ability to maintain somatic health, assessed as telomere attrition. Results We found substantial telomere attrition in transgenic fish, while maternal half-sibs growing at a lower, wild-type rate seemed better able to maintain the length of their telomeres during the same time period. Conclusions Our results are consistent with a trade-off between rapid growth and somatic (telomere) maintenance in growth-manipulated fish. Since telomere erosion reflects cellular ageing, our findings also support theories of ageing postulating that unrepaired somatic damage is associated with senescence.

Background Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. Results Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. Conclusions TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1782-z) contains supplementary material, which is available to authorized users.

Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.

Substantial evidence from the animal kingdom shows that there is a trade-off between benefits and costs associated with rapid somatic growth. One would therefore expect growth rates under natural conditions to be close to an evolutionary optimum. Nevertheless, natural selection in many salmonid species appears to be toward larger size and earlier emergence from spawning redds, indicating a potential for increased growth rate to evolve. We tested how selection for genetic variants (growth hormone transgenic coho salmon, Oncorhynchus kisutch, with more than doubled daily growth rate potential relative to wild genotypes) depended on predator timing and food abundance during the early period of life (fry stage). In artificial redds, fry of the fast-growing genotypes showed a highly significant developmental shift, emerging from gravel nests approximately two weeks sooner, but with an 18.6% reduced survival, relative to wild-genotype fry. In seminatural streams, fry of the fast-growing genotypes suffered higher predation than those of wild genotypes when predators were present at the time of fry emergence, but this difference was less pronounced when food was scarce. In streams where predators were introduced after emergence, fry survived equally well regardless of food availability. Surviving fry grew faster in habitats provided with more food, and fast-growing genotypes also grew faster than wild genotypes when predators arrived late and food was abundant. Fewer fish migrated downstream past a waterfall when food availability was high and in the presence of predators, and wild-genotype fry were more likely to migrate than fry of the fast-growing genotypes. After being returned to the experimental streams after migration, fast-growing genotypes survived equally well as those of the same genotypes that did not migrate, whereas migrating wild genotypes experienced higher mortality relative to those of the same genotypes that did not migrate. Comparisons of growth rates between siblings retained under hatchery conditions and those from habitats with the fastest growth in the experimental stream revealed that growth rates were similar for wild genotypes in both environments, whereas the fast-growing genotypes in the streams only realized 90% of their growth potential. The present study has shown that a major shift in developmental timing can alter critical early stages affecting survival and can have a significant effect on fitness. Furthermore, ecological conditions such as food abundance and predation pressure can strongly influence the potential for fast-growing variants to survive under natural conditions. The large-scale removal of many predatory species around the world may augment the evolution of increased intrinsic growth rates in some taxa.

Two Y-chromosome DNA markers (a repetitive sequence, OtY1, and a single-copy marker, GH-Y) tightly linked to the sex-determination locus have been examined for their association with sexual development among 55 populations of Chinook salmon (Oncorhynchus tshawytscha) from the Yukon, British Columbia, Washington, Oregon, and Idaho. Normal linkage has been observed in 96.7% of 2478 individuals examined. Only five males (0.44%) were found lacking both markers (none from Canadian systems), and 14 females (1.04%) from US populations and two females (0.15%) from Canadian populations were found to possess both markers. Variants identified included weakly amplifying alleles for GH-Y and OtY1 and structural variants identified by Southern-blot analysis. The frequency of variants in males was more than 2-fold that in females, and males deficient in GH-Y were more common (3.6%) than males deficient in the repetitive OtY1 sequence (0.7%). Some individuals (of both sexes) possessed fewer copies of the OtY1 repeat than normal males, revealing molecular dynamics that alter Y-chromosome structure within and among populations. A population (Hanford Reach) previously reported as having a high incidence of females possessing the OtY1 marker, and suspected of being sex-reversed, was found to have normal sex-marker genotypes in the present study.

In order to best predict the consequences of interaction between domestic and wild fish populations, the physiological and genetic differences that have occurred during the domestication process should be understood. Through production of multiple generations (F1 and F2 backcrosses) of domestic and wild rainbow trout (Oncorhynchus mykiss) hybrids, the present study examines genetic changes that have occurred during domestication, as well as the action of these changes when introgressed back into a wild genome. A strong correlation observed between the proportion of domestic genes within the genotype and growth indicates that additive gene action is responsible for a large amount of the phenotypic variability.

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.

Transgenic coho salmon strains containing an all-salmon growth hormone (GH) gene construct (OnMTGH1) have been examined. The transgene utilized is comprised of the metallothionein-B promoter driving the expression of the type-I growth hormone gene from sockeye salmon. Transgene DNA is integrated into the salmon genome, but is transmitted at low frequency from founder transgenic animals consistent with late integration following microinjection resulting in mosaic animals, whereas subsequent generations transmit transgene DNA as a stable Mendelian trait. Different families established from separate founder animals yield lines with unique growth characteristics suggesting important site-of-integration effects on transgene expression. Growth enhancement of transgenic salmon is initiated early, with advanced hatch timing but occurring also throughout the life history, particularly during the early phase in fresh water. GH transgenic fish showed precocious smoltification and onset of sexual maturation, but approximately normal adult body size, indicating that compression of the normal coho salmon life history is occurring. The viability of diploid GH transgenic salmon ranges from reductions to greater than that of controls among strains, and triploid transgenic animals had normal viability relative to diploid transgenic salmon. Triploid transgenic salmon display a reduction in growth rate relative to transgenic diploids, but are still significantly growth enhanced compared with nontransgenic controls. The distinct phenotypic characteristics of GH transgenic families suggest that evaluation for aquaculture and for risk assessments requires examination of strains on a case-by-case basis. Furthermore, strong effects of size at maturity in culture conditions were observed for nontransgenic wild strain coho salmon which were not apparent in GH transgenic salmon, indicating that strong genotype by environment effects strongly influence phenotype and complicate risk assessments.

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non-transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non-transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non-transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth-accelerated coho salmon.

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

A PCR-based method for the identification of genetically modified coho salmon (Oncorhynchus kisutch) has been developed and evaluated by a collaborative study. The coho salmon contained an "all-salmon" gene-construct (OnMTGH1) consisting of the metallothionein-B-promoter fused to the full-length type-1 growth hormone gene, both from sockeye salmon (Oncorhynchus nerka).

Growth hormone (GH) transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination) has not been fully ascertained. Hence, salmon were reared in large (350,000 L), semi-natural, seawater tanks (termed mesocosms) designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic salmon are capable of successful spawning, and can reproduce with wild-type fish from natural systems.

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.

Historically, the taxonomy and nomenclature of Japanese salmon have been in a state of confusion. Masu, amago and biwa salmon have been variously classified as distinct species, subspecies, or often conflicting or overlapping combinations of the two. In particular, the taxonomy of masu and amago salmon is obscured by their similarity in ecological and morphological traits. Here, DNA sequence analyses of nuclear and mitochondrial DNA were applied to clarify the genetic relationship between masu and amago salmon. No fixed differences were detected in the mitochondrial ND3 gene and control (D-loop) region, or in the nuclear growth hormone type-2 gene (GH2). However, the frequency of single nucleotide substitution alleles within GHZ intron C and size variants at a microsatellite locus nested within intron D differed markedly, providing genetic evidence to support a taxonomic distinction between the two types. The genetic data were related to previous mitochondrial DNA sequence analyses and alternativ...

The taxonomy of the PKX myxosporean, cause of proliferative kidney disease (PKD) of salmonids, has long been an enigma because only immature spores are consistently associated with the infection. In the early 1990s, one of us (M.L.K.) proposed that PKX may be the extrasporogonic form of Sphaerospora oncorhynchi, which has been observed in salmonids where PKD is enzootic. Recently, the sequence of the small subunit ribosomal DNA (SSU rDNA) of PKX was deposited in GenBank (U70623). We designed PKX-specific primers by aligning this sequence with that of coho salmon Oncorhynchus kisutch and with those of other myxosporeans. With these primers, a portion of the SSU rDNA of PKX was amplified and sequenced from the kidneys of infected salmonids from British Columbia, Washington, California, Newfoundland, and England. The maximum variation in sequence across 764 base pairs (bp) among all PKX isolates, including the original sequence (GenBank U70623), was 0.8%. Results indicated that PKX isolates from dif...

Rules of zoological nomenclature, morphological data, and ribosomal DNA sequence data support the validity of the genus Nucleospora, and its placement in the family Enterocytozoonidae. Although Nucleospora exhibits most of the distinguishing morphological characteristics of the family Enterocytozoonidae Cali and Owen, 1990, the distinctively different hosts (fish and humans, respectively) and sites of development (the nuclei of immature blood cells and the cytoplasm of enterocytes) support the placement of Nucleospora and Enterocytozoon into separate genera. Ribosomal DNA sequence comparisons between Nucleospora salmonis and Enterocytozoon bieneusi showed 19.8% genetic divergence in the large and small subunit regions. Although more inter- and intrageneric comparisons are needed before percent homology of ribosomal DNA can be used as a criterion for the separation of genera, the genetic divergence between the two species is sufficiently large to deter suppression of the genus Nucleospora as a junior synonym of Enterocytozoon. A polymerase chain reaction test for the detection of N. salmonis in chinook salmon (Oncorhynchus tshawytscha), based on N. salmonis-specific ribosomal DNA sequence, is described.

Transmission of the opAFPGHc gene construct from parental transgenic coho salmon to F 1 progeny has been observed. Just prior to first feeding, these offspring were found to fall into two distinct phenotypic classes on the basis of morphology and external colouration. One group possessed the normal brown colouration typical of coho salmon alevins, whereas the other had a distinct green colouration and showed signs of cranial deformities and opercular overgrowth. Polymerase chain reaction analysis revealed that green phenotype was correlated with the presence of the opAFPGHc gene construct, and thus colouration could be used to identify transgenic progeny. On this basis, frequencies of transgene transmission to F 1 progeny from four individuals ranged from 2.2 to 18.9%, while a fifth male produced no transgenic progeny. Prior to first feeding, the transgenic progeny were found to be 21.2% heavier and 11.9% longer than their non-transgenic siblings, suggesting that the expression of GH in early development can influence the rate or efficiency of conversion of yolk energy reserves. After 1 year of development of F 1 progeny, the atypical phenotype associated with overgrowth of cartilage in the cranial and opercular regions became progressively more severe and resulted in reduced viability.

DNA fingerprinting is a molecular biological technique that is widely used for identifying parentage and relatedness in plants and animals. To identify new DNA fingerprinting probes for use with salmonids, Southern blots of genomic DNA from chinook salmon (Oncorhynchus tshawytscha) were hybridized at low stringencies with 12 different oligonucleotides designed from published core sequences of variable number of tendem repeats. Seven of the 12 oligonucleotides produced highly variable fingerprint-like patterns; however, only 3 of these had clear, distinct bands. The estimated heterozygosity for one population of chinook salmon using the three oligonucleotides as probes ranged from 0.64 to 0.77. Those three oligonucleotides were further hybridized with DNA from two unrelated individuals from six other species of salmonids. A single-locus DNA fingerprint probe originally developed for chinook salmon was also hybridized with DNA from the other six species at moderate stringency. There were differences in the complexity and signal strength of the resulting banding pattern between species for a given probe. Estimates of variability (heterozygosity and band sharing) for the three oligonucleotide probes and OtSL1 were high, indicating that the probes were potentially useful genetic markers. The availability of these additional DNA fingerprint probes should assist in ecological and evolutionary studies in salmonids, as well as in efforts to estimate genetic diversity of populations.

Two single locus Variable Number Tandem Repeat (VNTR) DNA probes were used to test for differences in allele distribution between precociously mature male and immature chinook salmon, Oncorhynchus tshawytscha. Two populations were examined: Robertson Creek (RC) adult salmon, and Nicola River (NR) freshwater juveniles, or parr. Genomic DNA was extracted from 74 RC precociously mature adult males ('jacks') and 94 RC immature adults of the same age and from 45 NR precociously mature parr and 51 NR nonmaturing parr. The genomic DNA was hybridized with a single locus VNTR probe developed for chinook salmon (OtSL1), as well as one developed for Atlantic salmon, Salmo salar (Ssa1). The allele frequency distributions at both loci were significantly different for the RC jacks and immature fish, indicating a family effect on the incidence of precocious maturation in that population. No difference was found between the allele frequency distribution of the NR precocious and immature parr. A bin width sensitivity analysis showed that the comparisons of the allele frequency distributions were insensitive to the choice of bin size. No differences in heterozygosity were found between mature and immature fish at either locus for both stocks. Preliminary testing for family effects on phenotypes of interest, such as alternative life history strategies, can be performed using hypervariable VNTR DNA probes, prior to implementing costly and involved breeding programmes.

A simple procedure is described that is capable of determining the genetic sex of chinook salmon ( Oncorhynchus tshawytscha ). The test was developed by sequencing a Y-chromosomal DNA probe (OtY1) from this species and designing oligonucleotides for use in polymerase chain reactions (PCR). The PCR produces a male-specific fragment of 209 bp and several other non-sex-specific fragments of higher molecular weight. The pattern obtained from the PCR suggests that the Y-chromosome-specific fragments are repeated in the genome, consitent with previous observations made with Southern blot analysis. The assay can be rapidly and reliably performed on small pieces of tissue or a small drop of blood, thus obviating the need to sacrifice the animal. The assay for Y-chromosomal DNA was used to determine the genetic sex of precociously mature, androgen-treated chinook salmon. Both XX males and regular XY males were identified. Progeny obtained from these and control crosses showed that, as expected, fish positive for Y-chromosomal DNA produced both males and females, whereas those appearing negative for Y-chromosomal DNA produced only females. The application of such simple tests for Y-chromosomal DNA in fish should facilitate the development of monosex stocks for aquacultural purposes.

Genetic, environmental and interaction effects on the incidence of jacking in Oncorhynchus tshawytscha (chinook salmon)

Background Somatic growth is a complex process that involves the action and interaction of genes and environment. A number of quantitative trait loci (QTL) previously identified for body weight and condition factor in rainbow trout (Oncorhynchus mykiss), and two other salmonid species, were used to further investigate the genetic architecture of growth-influencing genes in this species. Relationships among previously mapped candidate genes for growth and their co-localization to identified QTL regions are reported. Furthermore, using a comparative genomic analysis of syntenic rainbow trout linkage group clusters to their homologous regions within model teleost species such as zebrafish, stickleback and medaka, inferences were made regarding additional possible candidate genes underlying identified QTL regions. Results Body weight (BW) QTL were detected on the majority of rainbow trout linkage groups across 10 parents from 3 strains. However, only 10 linkage groups (i.e., RT-3, -6, -8, -9, -10, -12, -13, -22, -24, -27) possessed QTL regions with chromosome-wide or genome-wide effects across multiple parents. Fewer QTL for condition factor (K) were identified and only six instances of co-localization across families were detected (i.e. RT-9, -15, -16, -23, -27, -31 and RT-2/9 homeologs). Of note, both BW and K QTL co-localize on RT-9 and RT-27. The incidence of epistatic interaction across genomic regions within different female backgrounds was also examined, and although evidence for interaction effects within certain QTL regions were evident, these interactions were few in number and statistically weak. Of interest, however, was the fact that these predominantly occurred within K QTL regions. Currently mapped growth candidate genes are largely congruent with the identified QTL regions. More QTL were detected in male, compared to female parents, with the greatest number evident in an F1 male parent derived from an intercross between domesticated and wild strain of rainbow trout which differed strongly in growth rate. Conclusions Strain background influences the degree to which QTL effects are evident for growth-related genes. The process of domestication (which primarily selects faster growing fish) may largely reduce the genetic influences on growth-specific phenotypic variation. Although heritabilities have been reported to be relatively high for both BW and K growth traits, the genetic architecture of K phenotypic variation appears less defined (i.e., fewer major contributing QTL regions were identified compared with BW QTL regions).