Benjamin F. Koop

Sequencing the genome of the Atlantic salmon (Salmo salar)

The International Collaboration to Sequence the Atlantic Salmon Genome (ICSASG) will produce a genome sequence that identifies and physically maps all genes in the Atlantic salmon genome and acts as a reference sequence for other salmonids.

The Sex Determining Loci and Sex Chromosomes in the Family Salmonidae

Salmonids are descended from a common ancestor that underwent an autotetraploidization event. After a whole genome duplication species could deal with sex determination by deleting one copy of SEX, the sex determining locus, or by recruiting a duplicated transcription factor to become a novel sex determining gene. It is not known which if any of these strategies salmonids adopted, but it appears that they all have primarily a genetic mechanism of sex determination with male heterogamety. The sharing of sex-linked markers on the X and Y chromosomes and the difficulty in identifying Y-specific markers indicate that X and Y chromosomes in salmonids have a large pseudoautosomal region and a small sex determining region. Linkage analyses suggest that either SEX differs in different lineages or else has remained the same and moved by transposition to different chromosomes. The identification of the sex chromosomes in salmonid species has not resolved this issue. It is clear that salmonids are at an early stage in sex chromosome differentiation and therefore provide a wonderful opportunity to study the evolution of sex determination. The availability of a reference salmonid genome sequence would provide an important resource for research in this area.

GO Trimming: Systematically reducing redundancy in large Gene Ontology datasets

BackgroundThe increased accessibility of gene expression tools has enabled a wide variety of experiments utilizing transcriptomic analyses. As these tools increase in prevalence, the need for improved standardization in processing and presentation of data increases, as does the need to guard against interpretation bias. Gene Ontology (GO) analysis is a powerful method of interpreting and summarizing biological functions. However, while there are many tools available to investigate GO enrichment, there remains a need for methods that directly remove redundant terms from enriched GO lists that often provide little, if any, additional information.FindingsHere we present a simple yet novel method called GO Trimming that utilizes an algorithm designed to reduce redundancy in lists of enriched GO categories. Depending on the needs of the user, this method can be performed with variable stringency. In the example presented here, an initial list of 90 terms was reduced to 54, eliminating 36 largely redundant terms. We also compare this method to existing methods and find that GO Trimming, while simple, performs well to eliminate redundant terms in a large dataset throughout the depth of the GO hierarchy.ConclusionsThe GO Trimming method provides an alternative to other procedures, some of which involve removing large numbers of terms prior to enrichment analysis. This method should free up the researcher from analyzing overly large, redundant lists, and instead enable the concise presentation of manageable, informative GO lists. The implementation of this tool is freely available at: http://lucy.ceh.uvic.ca/go_trimming/cbr_go_trimming.py

High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

BackgroundTwo decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response.ResultsGranulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-β (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of TH1 T cell lineage (IFN-γ, CD4) or TH2 (GATA-3) responses were differentially expressed.ConclusionsGranulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-β, IL-17A and its receptor suggests an involvement of TH17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a TH2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar)

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory‐related genes in the olfactory rosettes of different life stages in two anadromous and one non‐anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non‐anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory‐related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.

Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire.

BackgroundThe genomes of salmonids are considered pseudo-tetraploid undergoing reversion to a stable diploid state. Given the genome duplication and extensive biological data available for salmonids, they are excellent model organisms for studying comparative genomics, evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes. The evolution of the tetrapod hemoglobin genes is well studied; however, little is known about the genomic organization and evolution of teleost hemoglobin genes, particularly those of salmonids. The Atlantic salmon serves as a representative salmonid species for genomics studies. Given the well documented role of hemoglobin in adaptation to varied environmental conditions as well as its use as a model protein for evolutionary analyses, an understanding of the genomic structure and organization of the Atlantic salmon α and β hemoglobin genes is of great interest.ResultsWe identified four bacterial artificial chromosomes (BACs) comprising two hemoglobin gene clusters spanning the entire α and β hemoglobin gene repertoire of the Atlantic salmon genome. Their chromosomal locations were established using fluorescence in situ hybridization (FISH) analysis and linkage mapping, demonstrating that the two clusters are located on separate chromosomes. The BACs were sequenced and assembled into scaffolds, which were annotated for putatively functional and pseudogenized hemoglobin-like genes. This revealed that the tail-to-tail organization and alternating pattern of the α and β hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr β globin genes, than the genomes of other teleosts that have been sequenced.ConclusionsWe suggest that the most parsimonious evolutionary path leading to the present organization of the Atlantic salmon hemoglobin genes involves the loss of a single hemoglobin gene cluster after the whole genome duplication (WGD) at the base of the teleost radiation but prior to the salmonid-specific WGD, which then produced the duplicated copies seen today. We also propose that the relatively high number of hemoglobin genes as well as the presence of non-Bohr β hemoglobin genes may be due to the dynamic life history of salmon and the diverse environmental conditions that the species encounters.Data deposition: BACs S0155C07 and S0079J05 (fps135): GenBank GQ898924; BACs S0055H05 and S0014B03 (fps1046): GenBank GQ898925

A 44K microarray dataset of the changing transcriptome in developing Atlantic salmon (Salmo salar L.)

BackgroundAtlantic salmon (Salmo salar L.) is an environmentally and economically important organism and its gene content is reasonably well characterized. From a transcriptional standpoint, it is important to characterize the changes in gene expression over the course of unperturbed early development, from fertilization through to the parr stage.FindingsS. salar samples were taken at 17 time points from 2 to 89 days post fertilization. Total RNA was extracted and cRNA was synthesized and hybridized to a newly developed 44K oligo salmonid microarray platform. Quantified results were subjected to preliminary data analysis and submitted to NCBIs Gene Expression Omnibus (GEO). Data can be found under the GEO accession number GSE25938. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25938ConclusionsThroughout the entire period of development, several thousand genes were found to be differentially regulated. This work represents the trancriptional characterization of a very large geneset that will be extremely valuable in further examination of the transcriptional changes in Atlantic salmon during the first few months of development. The expression profiles can help to annotate salmon genes in addition to being used as references against any number of experimental variables to which developing salmonids might be subjected.

Identification of olfactory receptor genes in Atlantic salmon Salmo salar

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. The key components of the molecular pathways involved in imprinting and homing, however, are still unknown. Aquatic chemical cues are received through the nares and into the nasal cavity that contains a single olfactory organ, the olfactory rosette. The olfactory rosette contains sensory neurons, each of which is thought to express only one olfactory receptor. If odorants are involved in salmonid homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, to understand the molecular basis for imprinting and homing in Atlantic salmon Salmo salar it is important to identify and characterize the repertoire of olfactory receptors in this species. The first public assembly of the S. salar genome was searched for genes encoding three of the superfamilies of fish olfactory receptors: V2R-like (olfc), V1R-like (ora) and main olfactory receptor (mor). A further six ora genes were added to ora1 and ora2, which had been described previously. In addition, 48 putative mors were identified, 24 of which appear to be functional based on their gene structures and predicted amino-acid sequences. Phylogenetic analyses were then used to compare these S. salar olfactory receptor genes with those of zebrafish Danio rerio, two pufferfish species Takifugu rubripes and Tetraodon nigroviridis, medaka Oryzias latipes and three-spined stickleback Gasterosteus aculeatus.

Grayling (Thymallinae) phylogeny within salmonids: complete mitochondrial DNA sequences of Thymallus arcticus and Thymallus thymallus

The phylogenetic relationships among the three subfamilies (Salmoninae, Coregoninae and Thymallinae) in the Salmonidae have not been addressed extensively at the molecular level. In this study, the whole mitochondrial genomes of two Thymallinae species, Thymallus arcticus and Thymallus thymallus were sequenced, and the published mitochondrial genome sequences of other salmonids were used for Bayesian and maximum-likelihood phylogenetic analyses. These results support an ancestral Coregoninae, branching within the Salmonidae, with Thymallinae as the sister group to Salmoninae.

Identification of the Sex Chromosomes of Brown Trout (Salmo trutta) and Their Comparison with the Corresponding Chromosomes in Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss)

Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.