Aggressive behavior, brain size and domestication in clonal rainbow trout lines.

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

Antipredator behavior QTL: differences in rainbow trout clonal lines derived from wild and hatchery populations.

Variation in antipredator behavior may partially explain the survival differences seen between wild and hatchery trout and salmon. Antipredator behavior is thought to change during the domestication process, along with other traits. Investigations of antipredator behavior could benefit conservation efforts and supplementation programs. Our goal was to characterize the antipredator behavior in clonal rainbow trout lines derived from either wild or hatchery populations and identify genetic loci associated with variation between lines. We identified several behaviors that varied between clonal lines and QTL for several behavioral and size traits. Characterizing genetic variation underlying these behaviors may prove valuable in future conservation efforts by enabling monitoring of allele frequencies of loci affecting predation in wild populations.

A conserved haplotype controls parallel adaptation in geographically distant salmonid populations.

Salmonid fishes exhibit extensive local adaptations owing to abundant environmental variation and precise natal homing. This extensive local adaptation makes conservation and restoration of salmonids a challenge. For example, defining unambiguous units of conservation is difficult, and restoration attempts often fail owing to inadequate adaptive matching of translocated populations. A better understanding of the genetic architecture of local adaptation in salmonids could provide valuable information to assist in conserving and restoring natural populations of these important species. Here, we use a combination of laboratory crosses and next-generation sequencing to investigate the genetic architecture of the parallel adaptation of rapid development rate in two geographically and genetically distant populations of rainbow trout (Oncorhynchus mykiss). Strikingly, we find that not only is a parallel genetic mechanism used but that a conserved haplotype is responsible for this intriguing adaptation. The repeated use of adaptive genetic variation across distant geographical areas could be a general theme in salmonids and have important implications for conservation and restoration.

Differential gene expression in male and female rainbow trout embryos prior to the onset of gross morphological differentiation of the gonads.

BACKGROUND: There are large differences between the sexes at the genetic level; these differences include heterogametic sex chromosomes and/or differences in expression of genes between the sexes. In rainbow trout (Oncorhynchus mykiss) qRT-PCR studies have found significant differences in expression of several candidate sex determining genes. However, these genes represent a very small fraction of the genome and research in other species suggests there are large portions of the transcriptome that are differentially expressed between the sexes. These differences are especially noticeable once gonad differentiation and maturation has occurred, but less is known at earlier stages of development. Here we use data from a microarray and qRT-PCR to identify genes differentially expressed between the sexes at three time points in pre-hatch embryos, prior to the known timing of sexual differentiation in this species. RESULTS: The microarray study revealed 883 differentially expressed features between the sexes with roughly equal numbers of male and female upregulated features across time points. Most of the differentially expressed genes on the microarray were not related to sex function, suggesting large scale differences in gene expression between the sexes are present early in development. Candidate gene analysis revealed sox9, DMRT1, Nr5a1 and wt1 were upregulated in males at some time points and foxl2, ovol1, fst and cyp19a1a were upregulated in females at some time points. CONCLUSION: This is the first study to identify sexual dimorphism in expression of the genome during embryogenesis in any fish and demonstrates that transcriptional differences are present before the completion of gonadogenesis.

A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout.

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

A first generation BAC-based physical map of the rainbow trout genome.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. A bacterial artificial chromosome (BAC) physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. RESULTS: The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF) method. The clones were assembled into physical map contigs using the finger-printing contig (FPC) program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1) comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2) anchoring large contigs to the microsatellite-based genetic linkage map. CONCLUSION: The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for economically important traits and the incorporation of MAS into rainbow trout breeding programs.

The genetic basis of smoltification-related traits in Oncorhynchus mykiss.

The timing and propensity for migration between fresh- and seawater is a key theme in the diversity of life histories within the salmonid fishes. Across salmonid species, life-history strategies range from wholly freshwater-resident populations, to migratory and nonmigratory variation within populations, to populations and species that are primarily migratory. Despite the central theme of migration to the evolution of these fishes, the genetic architecture of migration-related processes is poorly understood. Using a genetic cross of clonal lines derived from migratory and nonmigratory life-history types of Onchorhynchus mykiss (steelhead and rainbow trout, respectively), we have dissected the genetic architecture of the complex physiological and morphological transformation that occurs immediately prior to seaward migration (termed smoltification). Quantitative trait loci (QTL) analyses were used to identify the number, effects, and genomic location of loci associated with smoltification-related traits, including growth and condition factor, body coloration, morphology, and osmoregulatory enzymes during the smoltification period. Genetic analyses revealed numerous QTL, but one locus in particular is associated with multiple traits in single and joint analyses. Dissecting the genetic architecture of this highly complex trait has profound implications for understanding the genetic and evolutionary basis of life-history diversity within and among migratory fishes.

Transcriptional profiling of MHC class I genes in rainbow trout infected with infectious hematopoietic necrosis virus.

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

Quantitative trait loci x maternal cytoplasmic environment interaction for development rate in Oncorhynchus mykiss.

Effects of maternal cytoplasmic environment (MCE) on development rate in rainbow trout were evaluated within a quantitative trait loci (QTL) analysis framework. Previous research had identified QTL for development rate in doubled haploid (DH) progeny produced from a cross between the Oregon State University (OSU) and the Swanson (SW) River rainbow trout clonal lines. In this study, progeny for QTL mapping were produced from a cross between the OSU and Clearwater (CW) River clonal lines. Doubled haploids were produced from the OSU x CW F1 by androgenesis using eggs from different females (or MCEs); with androgenesis, the maternal nuclear genome was destroyed by irradiation and diploidy was restored by blocking the first embryonic cleavage by heat shock. All embryos were incubated at the same temperature and development rate quantified as time to hatch. Using a linkage map constructed primarily with AFLP markers, QTL mapping was performed, including MCE covariates and QTL x MCE effects in models for testing. The major QTL for development rate in the OSU x SW cross overlaps with the major QTL found in this OSU x CW cross; effects at this locus were the same across MCEs. Both MCE and QTL x MCE effects contribute to variability in development rate, but QTL x MCE were minor and detected only at small-effect QTL.

A resource of single-nucleotide polymorphisms for rainbow trout generated by restriction-site associated DNA sequencing of doubled haploids.

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of a genome duplication event that occurred between 25 and 100 Ma. This situation complicates single-nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and not simple allelic variants. To differentiate PSVs from simple allelic variants, we used 19 homozygous doubled haploid (DH) lines that represent a wide geographical range of rainbow trout populations. In the first phase of the study, we analysed SbfI restriction-site associated DNA (RAD) sequence data from all the 19 lines and selected 11 lines for an extended SNP discovery. In the second phase, we conducted the extended SNP discovery using PstI RAD sequence data from the selected 11 lines. The complete data set is composed of 145,168 high-quality putative SNPs that were genotyped in at least nine of the 11 lines, of which 71,446 (49%) had minor allele frequencies (MAF) of at least 18% (i.e. at least two of the 11 lines). Approximately 14% of the RAD SNPs in this data set are from expressed or coding rainbow trout sequences. Our comparison of the current data set with previous SNP discovery data sets revealed that 99% of our SNPs are novel. In the support files for this resource, we provide annotation to the positions of the SNPs in the working draft of the rainbow trout reference genome, provide the genotypes of each sample in the discovery panel and identify SNPs that are likely to be in coding sequences.

Transcriptome profiling of embryonic development rate in rainbow trout advanced backcross introgression lines.

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

Transcriptome profiles of livers and kidneys from three rainbow trout (Oncorhynchus mykiss) clonal lines distinguish stocks from three allopatric populations.

Transcriptome profiling is a powerful means of simultaneously identifying large numbers of genes that respond transcriptionally to stimuli of any sort. Whereas individuality at the level of genomic sequence is readily revealed and can be expected to influence transcriptional responses, knowledge of the global transcriptomic consequences of genomic individuality is in its infancy. Appreciation of the inherent variability of biological systems gives us confidence in predicting that no two individuals in any outbred population will respond identically to a stimulus. More critical for comparative studies, even unstimulated transcriptomes will be distinctive for each individual. To assess the confidence with which inferences may be drawn from transcriptome profiling when genetically identical samples can be assured, we examined the unprovoked transcriptomes of hepatic and pronephric (head kidney) tissues in three clonal lines of Rainbow trout (Oncorhynchus mykiss). Clonal individuals derived from three allopatric populations presented transcriptional profiles for both liver and pronephros that were not statistically significantly different within each clonal line; however each clonal line was distinguished by a subset of genes with constitutively different transcript abundance. Among these, immunologically-relevant genes were over-represented, possibly reflecting evolutionarily recent, pathogen-driven genetic sweeps.

Behavioral differences among rainbow trout clonal lines.

Salmonid fish show considerable geographical variation in morphology, physiology, and behavior. Understanding the genetic mechanisms underlying this variation could be useful for enhancing aquaculture stocks, identifying unique populations for conservation, and determining the genetic factors underlying natural adaptation and domestication. As a first step toward the genetic dissection of salmonid behavioral diversity, we examined variation in behavior patterns among four clonal lines of rainbow trout ( Oncorhynchus mykiss ) derived from geographically diverse source populations with different domestication histories. Clonal lines were crossed with two outbred (i.e., not homozygous) females, and the resulting progenies were reared and tested under identical conditions. Clonal line had significant genetic effects on mean swim level, hiding, foraging, startle response, and aggression level. Multiple comparisons suggest that domestication history of the source populations had a strong influence on these behavior patterns. Progeny of two clonal lines derived from populations reared in captivity for over 100 years exhibited reductions in predator avoidance behavior patterns and increases in aggression compared to progeny of two clonal lines from more recently domesticated populations. These results will facilitate future investigation of the genetic factors underlying population variation in these behavior patterns influenced by domestication.

Polymorphism and differentiation of rainbow trout Y chromosomes.

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.