Joseph P. Brunelli

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.

Y-specific sequences and polymorphisms in rainbow trout and Chinook salmon

Improved methods for genetically sexing salmonids and for characterization of Y-chromosome homologies between species can contribute to understanding the evolution of sex chromosomes and sex-determining mechanisms. In this study we have explored 12.5 kb of Y-chromosome-specific sequence flanking the previously described OtY2 locus in Chinook salmon (Oncorhynchus tshawytscha) and 21 kb of homologous rainbow trout (Oncorhynchus mykiss) Y-chromosome-specific sequence. This is the first confirmed Y-specific sequence for rainbow trout. New Y-specific markers are described for Chinook salmon (OtY3) and rainbow trout (OmyY1), which are readily detected by PCR assays and are advantageous because they also produce autosomal control amplification products. Additionally, AFLP analysis of Chinook salmon yielded another potential Y-chromosome marker. These descriptions will facilitate genotypic sexing and should be useful for population studies of Y-chromosome polymorphisms and for future studies to characterize what appears to be a common sex-determining mechanism between these species.

A New Y-Chromosome-Specific Marker for Pacific Salmon

Abstract A genetic marker showing Y-chromosome-specific linkage in Chinook salmon Oncorhynchus tshawytscha, chum salmon O. keta, and coho salmon O. kisutch has been recovered by amplified fragment length polymorphism screening of haploid Chinook salmon embryos produced by androgenesis. Nested polymerase chain reaction primers designed from this sequence show sex-specific amplification. This region includes an apparent pseudogene of an open reading frame found autosomally in all Pacific salmonids tested. The test is not successful for rainbow trout O. mykiss or pink salmon O. gorbuscha but shows promise for use with sockeye salmon. Our approach should be applicable to isolating sex-specific sequences in other fish species. This marker (OtY2-WSU) should have applications in salmonid aquaculture, in studies of Y chromosome evolution, and in investigating possible sex reversal events in Pacific salmon.

Mapping of five candidate sex-determining loci in rainbow trout (Oncorhynchus mykiss)

BackgroundRainbow trout have an XX/XY genetic mechanism of sex determination where males are the heterogametic sex. The homology of the sex-determining gene (SDG) in medaka to Dmrt1 suggested that SDGs evolve from downstream genes by gene duplication. Orthologous sequences of the major genes of the mammalian sex determination pathway have been reported in the rainbow trout but the map position for the majority of these genes has not been assigned.ResultsFive loci of four candidate genes (Amh, Dax1, Dmrt1 and Sox6) were tested for linkage to the Y chromosome of rainbow trout. We exclude the role of all these loci as candidates for the primary SDG in this species. Sox6i and Sox6ii, duplicated copies of Sox6, mapped to homeologous linkage groups 10 and 18 respectively. Genotyping fishes of the OSU × Arlee mapping family for Sox6i and Sox6ii alleles indicated that Sox6i locus might be deleted in the Arlee lineage.ConclusionAdditional candidate genes should be tested for their linkage to the Y chromosome. Mapping data of duplicated Sox6 loci supports previously suggested homeology between linkage groups 10 and 18. Enrichment of the rainbow trout genomic map with known gene markers allows map comparisons with other salmonids. Mapping of candidate sex-determining loci is important for analyses of potential autosomal modifiers of sex-determination in rainbow trout.

Heat shock protein (HSP70) RNA expression differs among rainbow trout (Oncorhynchus mykiss) clonal lines

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

Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA.

A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.

Identification of single nucleotide polymorphisms from the transcriptome of an organism with a whole genome duplication.

BackgroundThe common ancestor of salmonid fishes, including rainbow trout (Oncorhynchus mykiss), experienced a whole genome duplication between 20 and 100 million years ago, and many of the duplicated genes have been retained in the trout genome. This retention complicates efforts to detect allelic variation in salmonid fishes. Specifically, single nucleotide polymorphism (SNP) detection is problematic because nucleotide variation can be found between the duplicate copies (paralogs) of a gene as well as between alleles.ResultsWe present a method of differentiating between allelic and paralogous (gene copy) sequence variants, allowing identification of SNPs in organisms with multiple copies of a gene or set of genes. The basic strategy is to: 1) identify windows of unique cDNA sequences with homology to each other, 2) compare these unique cDNAs if they are not shared between individuals (i.e. the cDNA is homozygous in one individual and homozygous for another cDNA in the other individual), and 3) give a “SNP score” value between zero and one to each candidate sequence variant based on six criteria. Using this strategy we were able to detect about seven thousand potential SNPs from the transcriptomes of several clonal lines of rainbow trout. When directly compared to a pre-validated set of SNPs in polyploid wheat, we were also able to estimate the false-positive rate of this strategy as 0 to 28% depending on parameters used.ConclusionsThis strategy has an advantage over traditional techniques of SNP identification because another dimension of sequencing information is utilized. This method is especially well suited for identifying SNPs in polyploids, both outbred and inbred, but would tend to be conservative for diploid organisms.

Single-Nucleotide Polymorphisms Associated with Allozyme Differences between Inland and Coastal Rainbow Trout

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

Deep divergence and apparent sex-biased dispersal revealed by a Y-linked marker in rainbow trout.

Y-chromosome and mitochondrial DNA markers can reveal phylogenetic patterns by allowing tracking of male and female lineages, respectively. We used sequence data from a recently discovered Y-linked marker and a mitochondrial marker to examine phylogeographic structure in the widespread and economically important rainbow trout (Oncorhynchus mykiss). Two distinct geographic groupings that generally correspond to coastal and inland subspecies were evident within the Y-marker network while the mtDNA haplotype network showed little geographic structure. Our results suggest that male-specific behavior has prevented widespread admixture of Y haplotypes and that gene flow between the coastal and inland subspecies has largely occurred through females. This new Y marker may also aid conservation efforts by genetically identifying inland populations that have not hybridized with widely stocked coastal-derived hatchery fish.

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.