Glenn A. Cooper

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

BackgroundWe have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested.ResultsWe have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA.ConclusionWe validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.

A salmonid EST genomic study: genes, duplications, phylogeny and microarrays

BackgroundSalmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish.Results298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species.ConclusionAn extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94–96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent with an ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, provide valuable new tools to study salmonids.

Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

BackgroundSalmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution.ResultsFrom existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates.Conclusions9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

Genomic organization of duplicated major histocompatibility complex class I regions in Atlantic salmon (Salmo salar)

BackgroundWe have previously identified associations between major histocompatibility complex (MHC) class I and resistance towards bacterial and viral pathogens in Atlantic salmon. To evaluate if only MHC or also closely linked genes contributed to the observed resistance we ventured into sequencing of the duplicated MHC class I regions of Atlantic salmon.ResultsNine BACs covering more than 500 kb of the two duplicated MHC class I regions of Atlantic salmon were sequenced and the gene organizations characterized. Both regions contained the proteasome components PSMB8, PSMB9, PSMB9-like and PSMB10 in addition to the transporter for antigen processing TAP2, as well as genes for KIFC1, ZBTB22, DAXX, TAPBP, BRD2, COL11A2, RXRB and SLC39A7. The IA region contained the recently reported MHC class I Sasa-ULA locus residing approximately 50 kb upstream of the major Sasa-UBA locus. The duplicated class IB region contained an MHC class I locus resembling the rainbow trout UCA locus, but although transcribed it was a pseudogene. No other MHC class I-like genes were detected in the two duplicated regions. Two allelic BACs spanning the UBA locus had 99.2% identity over 125 kb, while the IA region showed 82.5% identity over 136 kb to the IB region. The Atlantic salmon IB region had an insert of 220 kb in comparison to the IA region containing three chitin synthase genes.ConclusionWe have characterized the gene organization of more than 500 kb of the two duplicated MHC class I regions in Atlantic salmon. Although Atlantic salmon and rainbow trout are closely related, the gene organization of their IB region has undergone extensive gene rearrangements. The Atlantic salmon has only one class I UCA pseudogene in the IB region while trout contains the four MHC UCA, UDA, UEA and UFA class I loci. The large differences in gene content and most likely function of the salmon and trout class IB region clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa.

Identification of genes associated with heat tolerance in Arctic charr exposed to acute thermal stress

Arctic charr is an especially attractive aquaculture species given that it features the desirable tissue traits of other salmonids and is bred and grown at inland freshwater tank farms year round. It is of interest to develop upper temperature tolerant (UTT) strains of Arctic charr to increase the robustness of the species in the face of climate change and to enable production in more southern regions. We used a genomics approach that takes advantage of the well-studied Atlantic salmon genome to identify genes that are associated with UTT in Arctic charr. Specifically, we conducted an acute temperature trial to identify temperature tolerant and intolerant Arctic charr individuals, which were subject to microarray and qPCR analysis to identify candidate UTT genes. These were compared with genes annotated in a quantitative trait locus (QTL) region that was previously identified as associated with UTT in rainbow trout and Arctic charr and that we sequenced in Atlantic salmon. Our results suggest that small heat shock proteins as well as HSP-90 genes are associated with UTT. Furthermore, hemoglobin expression was significantly downregulated in tolerant compared with intolerant fish. Finally, QTL analysis and expression profiling identified COUP-TFII as a candidate UTT gene, although its specific role is unclear given the identification of two transcripts, which appear to have different expression patterns. Our results highlight the importance of using more than one approach to identify candidate genes, particularly when examining a complicated trait such as UTT in a highly complex genome for which there is no reference genome.

Evolution of duplicated IgH loci in Atlantic salmon, Salmo salar.

BackgroundThe Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. These duplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the IGH-A (1.1 Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region have been sequenced and analyzed.ResultsThe present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cμ region, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 VH segments which could be classified into 18 families. This is the largest number of VH families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B loci have evolved independently in the short time after the recent genome duplication approximately 60 mya.ConclusionsOur results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, Lepeophtheirus salmonis

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis.

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.

Functional adaptive diversity of the Atlantic salmon T-cell receptor gamma locus.

The Atlantic salmon (Salmo salar) T-cell receptor gamma (TCRgamma) gene has extensive diversity in its capacity for antigen recognition due to the V-J-C gene segments recombinational possibilities, and N-region diversity. This is the first report completely characterizing and annotating a TCRgamma gene locus in teleosts. We identified two different TCRgamma loci in Atlantic salmon. The first locus, TCRgamma 1, spans 260 kbp and contains four tandemly repeated clusters each of which consists of 1-4 variable (V) segments, 1-2 sets of a joining (J) segment and a constant (C) region. In total, 10 V segments, 5 J segments and 5 C regions were found in locus 1. In the second locus, TCRgamma 2, a single non-expressed V-J-C cluster was found. Surprisingly, the Atlantic salmon TCRgamma loci have a larger number of C regions relative to other teleosts, mouse and human. In addition, each Atlantic salmon TCRgamma C region has a different connecting peptide region that may result in distinct cellular responses. Expression data confirm the diverse repertoire found at the genomic level. At least 6 out of 7 functional V segments, all 5 J segments and all 5 C regions found in TCRgamma 1 were identified in TCRgamma transcription analysis. The identification and characterization of the functional TCRgamma 1 along with the associated TCR alpha/delta locus suggest that Atlantic salmon have a functional gammadelta T-cell immune component.

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.