Establishing analytical validity of BeadChip array genotype data by comparison to whole-genome sequence and standard benchmark datasets.

BACKGROUND: Clinical use of genotype data requires high positive predictive value (PPV) and thorough understanding of the genotyping platform characteristics. BeadChip arrays, such as the Global Screening Array (GSA), potentially offer a high-throughput, low-cost clinical screen for known variants. We hypothesize that quality assessment and comparison to whole-genome sequence and benchmark data establish the analytical validity of GSA genotyping. METHODS: To test this hypothesis, we selected 263 samples from Coriell, generated GSA genotypes in triplicate, generated whole genome sequence (rWGS) genotypes, assessed the quality of each set of genotypes, and compared each set of genotypes to each other and to the 1000 Genomes Phase 3 (1KG) genotypes, a performance benchmark. For 59 genes (MAP59), we also performed theoretical and empirical evaluation of variants deemed medically actionable predispositions. RESULTS: Quality analyses detected sample contamination and increased assay failure along the chip margins. Comparison to benchmark data demonstrated that > 82% of the GSA assays had a PPV of 1. GSA assays targeting transitions, genomic regions of high complexity, and common variants performed better than those targeting transversions, regions of low complexity, and rare variants. Comparison of GSA data to rWGS and 1KG data showed > 99% performance across all measured parameters. Consistent with predictions from prior studies, the GSA detection of variation within the MAP59 genes was 3/261. CONCLUSION: We establish the analytical validity of GSA assays using quality analytics and comparison to benchmark and rWGS data. GSA assays meet the standards of a clinical screen although assays interrogating rare variants, transversions, and variants within low-complexity regions require careful evaluation.

Expression analysis of sex-determining pathway genes during development in male and female Atlantic salmon (Salmo salar).

We studied the expression of 28 genes that are involved in vertebrate sex-determination or sex-differentiation pathways, in male and female Atlantic salmon (Salmo salar) in 11 stages of development from fertilization to after first feeding. Gene expression was measured in half-sibs that shared the same dam. The sire of family 1 was a sex-reversed female (i.e., genetically female but phenotypically male), and so the progeny of this family are all female. The sire of family 2 was a true male, and so the offspring were 50% male and 50% female. Gene expression levels were compared among three groups: 20 female offspring of the cross between a regular female and the sex-reversed female (family 1, first group), ∼ 10 females from the cross between a regular female and a regular male (family 2, second group) and ∼ 10 males from this same family (family 2, third group). Statistically significant differences in expression levels between males and the two groups of females were observed for two genes, gsdf and amh/mis, in the last four developmental stages examined. SdY, the sex-determining gene in rainbow trout, appeared to be expressed in males from 58 days postfertilization (dpf). Starting at 83 dpf, ovarian aromatase, cyp19a, expression appeared to be greater in both groups of females compared with males, but this difference was not statistically significant. The time course of expression suggests that sdY may be involved in the upregulation of gsdf and amh/mis and the subsequent repression of cyp19a in males via the effect of amh/mis.

Genomic Instability of the Sex-Determining Locus in Atlantic Salmon (Salmo salar).

Atlantic salmon and rainbow trout, like other members of the subfamily Salmoninae, are gonochoristic with male heterogamety. The finding that sex-linked genetic markers varied between species suggested that the sex-determining gene differs among salmonid species, or that there is one sex-determining gene that has the capacity to move around the genome. The discovery of sdY, the sex-determining gene in rainbow trout, and its presence in many male salmonids gave support to the latter. Additional evidence for a salmonid-specific, sex-determining jumping gene came from the mapping of the sex-determining locus to three different chromosomes in Tasmanian male Atlantic salmon lineages. To characterize the sex-determining region, we isolated three sdY containing BACs from an Atlantic salmon male library. Sequencing of these BACs yielded two contigs, one of which contained the sdY gene. Sequence analysis of the borders of male-specific and female/male common regions revealed highly repetitive sequences associated with mobile elements, which may allow an sdY cassette to jump around the genome. FISH analysis using a BAC or a plasmid containing the sdY gene showed that the sdY gene did indeed localize to the chromosomes where SEX had been mapped in different Tasmanian Atlantic salmon families. Moreover, the plasmid sdY gene probe hybridized primarily to one of the sex chromosomes as would be expected of a male-specific gene. Our results suggest that a common salmonid sex-determining gene (sdY) can move between three specific loci on chromosomes 2, 3, and 6, giving the impression that there are multiple SEX loci both within and between salmonid species.

Epithelial Cadherin Determines Resistance to Infectious Pancreatic Necrosis Virus in Atlantic Salmon.

Infectious pancreatic necrosis virus (IPNV) is the cause of one of the most prevalent diseases in farmed Atlantic salmon (Salmo salar). A quantitative trait locus (QTL) has been found to be responsible for most of the genetic variation in resistance to the virus. Here we describe how a linkage disequilibrium-based test for deducing the QTL allele was developed, and how it was used to produce IPN-resistant salmon, leading to a 75% decrease in the number of IPN outbreaks in the salmon farming industry. Furthermore, we describe how whole-genome sequencing of individuals with deduced QTL genotypes was used to map the QTL down to a region containing an epithelial cadherin (cdh1) gene. In a coimmunoprecipitation assay, the Cdh1 protein was found to bind to IPNV virions, strongly indicating that the protein is part of the machinery used by the virus for internalization. Immunofluorescence revealed that the virus colocalizes with IPNV in the endosomes of homozygous susceptible individuals but not in the endosomes of homozygous resistant individuals. A putative causal single nucleotide polymorphism was found within the full-length cdh1 gene, in phase with the QTL in all observed haplotypes except one; the absence of a single, all-explaining DNA polymorphism indicates that an additional causative polymorphism may contribute to the observed QTL genotype patterns. Cdh1 has earlier been shown to be necessary for the internalization of certain bacteria and fungi, but this is the first time the protein is implicated in internalization of a virus.

Detection of quantitative trait loci (QTL) related to grilsing and late sexual maturation in Atlantic salmon (Salmo salar).

In Atlantic salmon aquaculture, early sexual maturation represents a major problem for producers. This is especially true for grilse, which mature after one sea winter before reaching a desirable harvest weight, rather than after two sea winters. Salmon maturing as grilse have a much lower market value than later maturing individuals. For this reason, most companies desire fish that grow fast and mature late. Marker-assisted selection has the potential to improve the efficiency of selection against early maturation and for late sexual maturation; however, studies identifying age of sexual maturation-related genetic markers are lacking for Atlantic salmon. Therefore, we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype five families from the Mainstream Canada broodstock program and search for SNPs associated with early (grilsing) or late sexual maturation. There were 529 SNP loci that were variable across all five families, and this was the set that was used for quantitative trait loci (QTL) analysis. GridQTL identified two chromosomes, Ssa10 and Ssa21, containing QTL related to grilsing. In contrast, only one QTL, on Ssa18, was found linked to late maturation in Atlantic salmon. Our previous work on these five families did not identify genome-wide significant growth-related QTL on Ssa10, Ssa21, or Ssa18. Therefore, taken together, these results suggest that both grilsing and late sexual maturation are controlled independently of one another and also from growth-related traits. The identification of genomic regions associated with grilsing or late sexual maturation provide an opportunity to incorporate this information into selective breeding programs that will enhance Atlantic salmon farming.

Characterization of the Atlantic salmon (Salmo salar) brain-type fatty acid binding protein (fabp7) genes reveals the fates of teleost fabp7 genes following whole genome duplications.

Whole genome duplications (WGDs) are considered to have been a driving force in the generation of evolutionary diversity that is characteristic of higher eukaryotes. The ancestor of salmonids underwent two additional WGDs compared to mammals, one (3R) at the base of the teleost radiation and another (4R) in the common ancestor of extant salmonids. We have chosen the fatty acid binding protein (fabp) gene family as a model to study the fate of duplicated genes in teleosts following WGDs. As previously described for zebrafish, we identified two copies (fabp7a and fabp7b) of the brain-type fabp gene in several fish including rainbow smelt, but there was only a single transcript in northern pike, the closest relative of the salmonids, and two rather than the expected four fabp7 genes in Atlantic salmon, rainbow trout and grayling. A phylogenetic analysis revealed that a loss of the fabp7a gene occurred in the common ancestor of the northern pike and salmonids after it had diverged from the rainbow smelt, and that the 4R WGD then gave rise to the fabp7bI and fabp7bII observed in salmonids. This is supported by genetic mapping that placed the Atlantic salmon duplicated fabp7b genes on homeologous chromosomes. There was no evidence of neo-functionalization in the salmonid fabp7bI and fabp7bII genes based on dN/dS ratios and an examination of amino acid substitutions. Atlantic salmon fabp7bI and fabp7bII genes are both expressed broadly like fabp7b expression in northern pike. However, only Atlantic salmon fabp7bII, like its counterpart in northern pike and zebrafish, was expressed in the liver. A comparison of ~2000bp upstream of Atlantic salmon fabp7b gene duplicates revealed an insertion of 62bp in fabp7bI relative to fabp7bII. The presence of predicted transcription factor binding sites in this insertion sequence may explain the differential expression of the fabp7b gene duplicates in Atlantic salmon liver.

Gene duplication and divergence of long wavelength-sensitive opsin genes in the guppy, Poecilia reticulata.

Female preference for male orange coloration in the genus Poecilia suggests a role for duplicated long wavelength-sensitive (LWS) opsin genes in facilitating behaviors related to mate choice in these species. Previous work has shown that LWS gene duplication in this genus has resulted in expansion of long wavelength visual capacity as determined by microspectrophotometry (MSP). However, the relationship between LWS genomic repertoires and expression of LWS retinal cone classes within a given species is unclear. Our previous study in the related species, Xiphophorus helleri, was the first characterization of the complete LWS opsin genomic repertoire in conjunction with MSP expression data in the family Poeciliidae, and revealed the presence of four LWS loci and two distinct LWS cone classes. In this study we characterized the genomic organization of LWS opsin genes by BAC clone sequencing, and described the full range of cone cell types in the retina of the colorful Cumaná guppy, Poecilia reticulata. In contrast to X. helleri, MSP data from the Cumaná guppy revealed three LWS cone classes. Comparisons of LWS genomic organization described here for Cumaná to that of X. helleri indicate that gene divergence and not duplication was responsible for the evolution of a novel LWS haplotype in the Cumaná guppy. This lineage-specific divergence is likely responsible for a third additional retinal cone class not present in X. helleri, and may have facilitated the strong sexual selection driven by female preference for orange color patterns associated with the genus Poecilia.

Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family.

BACKGROUND: Several novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach. RESULTS: We have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX3C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX6C motif in Ig1 and a CX7C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX3C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs. CONCLUSIONS: This report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.

Comparative genomic analysis of Atlantic salmon, Salmo salar, from Europe and North America.

BACKGROUND: Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing of mtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from North America and Europe are genetically distinct. These observations are supported by karyotype analysis, which revealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29 pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this map with the well developed map for European Atlantic salmon. RESULTS: We used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAP mapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1) whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences in recombination rates between female and male Atlantic salmon have been noted, separate genetic maps were constructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male map has 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27 composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from the Western Atlantic. CONCLUSIONS: A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for the difference in the chromosome numbers between European and North American Atlantic salmon: Linkage groups AS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into a single NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it will require finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison of the genomic architecture of Atlantic salmon from North America and Europe with respect to chromosome organization.

Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire.

BACKGROUND: The 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 ß hemoglobin genes is of great interest. RESULTS: We identified four bacterial artificial chromosomes (BACs) comprising two hemoglobin gene clusters spanning the entire α and ß 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 ß hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr ß globin genes, than the genomes of other teleosts that have been sequenced. CONCLUSIONS: We 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 ß 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.

Genomic organization of duplicated short wave-sensitive and long wave-sensitive opsin genes in the green swordtail, Xiphophorus helleri.

BACKGROUND: Long wave-sensitive (LWS) opsin genes have undergone multiple lineage-specific duplication events throughout the evolution of teleost fishes. LWS repertoire expansions in live-bearing fishes (family Poeciliidae) have equipped multiple species in this family with up to four LWS genes. Given that color vision, especially attraction to orange male coloration, is important to mate choice within poeciliids, LWS opsins have been proposed as candidate genes driving sexual selection in this family. To date the genomic organization of these genes has not been described in the family Poeciliidae, and little is known about the mechanisms regulating the expression of LWS opsins in any teleost. RESULTS: Two BAC clones containing the complete genomic repertoire of LWS opsin genes in the green swordtail fish, Xiphophorus helleri, were identified and sequenced. Three of the four LWS loci identified here were linked in a tandem array downstream of two tightly linked short wave-sensitive 2 (SWS2) opsin genes. The fourth LWS opsin gene, containing only a single intron, was not linked to the other three and is the product of a retrotransposition event. Genomic and phylogenetic results demonstrate that the LWS genes described here share a common evolutionary origin with those previously characterized in other poeciliids. Using qualitative RT-PCR and MSP we showed that each of the LWS and SWS2 opsins, as well as three other cone opsin genes and a single rod opsin gene, were expressed in the eyes of adult female and male X. helleri, contributing to six separate classes of adult retinal cone and rod cells with average lambdamax values of 365 nm, 405 nm, 459 nm, 499 nm, 534 nm and 568 nm. Comparative genomic analysis identified two candidate teleost opsin regulatory regions containing putative CRX binding sites and hormone response elements in upstream sequences of LWS gene regions of seven teleost species, including X. helleri. CONCLUSIONS: We report the first complete genomic description of LWS and SWS2 genes in poeciliids. These data will serve as a reference for future work seeking to understand the relationship between LWS opsin genomic organization, gene expression, gene family evolution, sexual selection and speciation in this fish family.

Comprehensive analysis of MHC class I genes from the U-, S-, and Z-lineages in Atlantic salmon.

BACKGROUND: We have previously sequenced more than 500 kb of the duplicated MHC class I regions in Atlantic salmon. In the IA region we identified the loci for the MHC class I gene Sasa-UBA in addition to a soluble MHC class I molecule, Sasa-ULA. A pseudolocus for Sasa-UCA was identified in the nonclassical IB region. Both regions contained genes for antigen presentation, as wells as orthologues to other genes residing in the human MHC region. RESULTS: The genomic localisation of two MHC class I lineages (Z and S) has been resolved. 7 BACs were sequenced using a combination of standard Sanger and 454 sequencing. The new sequence data extended the IA region with 150 kb identifying the location of one Z-lineage locus, ZAA. The IB region was extended with 350 kb including three new Z-lineage loci, ZBA, ZCA and ZDA in addition to a UGA locus. An allelic version of the IB region contained a functional UDA locus in addition to the UCA pseudolocus. Additionally a BAC harbouring two MHC class I genes (UHA) was placed on linkage group 14, while a BAC containing the S-lineage locus SAA (previously known as UAA) was placed on LG10. Gene expression studies showed limited expression range for all class I genes with exception of UBA being dominantly expressed in gut, spleen and gills, and ZAA with high expression in blood. CONCLUSION: Here we describe the genomic organization of MHC class I loci from the U-, Z-, and S-lineages in Atlantic salmon. Nine of the described class I genes are located in the extension of the duplicated IA and IB regions, while three class I genes are found on two separate linkage groups. The gene organization of the two regions indicates that the IB region is evolving at a different pace than the IA region. Expression profiling, polymorphic content, peptide binding properties and phylogenetic relationship show that Atlantic salmon has only one MHC class Ia gene (UBA), in addition to a multitude of nonclassical MHC class I genes from the U-, S- and Z-lineages.

Assignment of Atlantic salmon (Salmo salar) linkage groups to specific chromosomes: conservation of large syntenic blocks corresponding to whole chromosome arms in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Most teleost species, especially freshwater groups such as the Esocidae which are the closest relatives of salmonids, have a karyotype comprising 25 pairs of acrocentric chromosomes and 48-52 chromosome arms. After the common ancestor of salmonids underwent a whole genome duplication, its karyotype would have 100 chromosome arms, and this is reflected in the modal range of 96-104 seen in extant salmonids (e.g., rainbow trout). The Atlantic salmon is an exception among the salmonids as it has 72-74 chromosome arms and its karyotype includes 12 pairs of large acrocentric chromosomes, which appear to be the result of tandem fusions. The purpose of this study was to integrate the Atlantic salmon's linkage map and karyotype and to compare the chromosome map with that of rainbow trout. RESULTS: The Atlantic salmon genetic linkage groups were assigned to specific chromosomes in the European subspecies using fluorescence in situ hybridization with BAC probes containing genetic markers mapped to each linkage group. The genetic linkage groups were larger for metacentric chromosomes compared to acrocentric chromosomes of similar size. Comparison of the Atlantic salmon chromosome map with that of rainbow trout provides strong evidence for conservation of large syntenic blocks in these species, corresponding to entire chromosome arms in the rainbow trout. CONCLUSION: It had been suggested that some of the large acrocentric chromosomes in Atlantic salmon are the result of tandem fusions, and that the small blocks of repetitive DNA in the middle of the arms represent the sites of chromosome fusions. The finding that the chromosomal regions on either side of the blocks of repetitive DNA within the larger acrocentric chromosomes correspond to different rainbow trout chromosome arms provides support for this hypothesis.

Genomic organization and evolution of the vomeronasal type 2 receptor-like (OlfC) gene clusters in Atlantic salmon, Salmo salar.

There are three major multigene superfamilies of olfactory receptors (OR, V1R, and V2R) in mammals. The ORs are expressed in the main olfactory organ, whereas the V1Rs and V2Rs are located in the vomeronasal organ. Fish only possess one olfactory organ in each nasal cavity, the olfactory rosette; therefore, it has been proposed that their V2R-like genes be classified as olfactory C family G protein-coupled receptors (OlfC). There are large variations in the sizes of OR gene repertoires. Previous studies have shown that fish have between 12 and 46 functional V2R-like genes, whereas humans have lost all functional V2Rs, and frog sp. have more than 240. Pseudogenization of V2R genes is a prevalent event across species. In the mouse and frog genomes, there are approximately double the number of pseudogenes compared with functional genes. An oligonucleotide probe was designed from a conserved sequence from four Atlantic salmon OlfC genes and used to screen the Atlantic salmon bacterial artificial chromosome (BAC) library. Hybridization-positive BACs were matched to fingerprint contigs, and representative BACs were shotgun cloned and sequenced. We identified 55 OlfC genes. Twenty-nine of the OlfC genes are classified as putatively functional genes and 26 as pseudogenes. The OlfC genes are found in two genomic clusters on chromosomes 9 and 20. Phylogenetic analysis revealed that the OlfC genes could be divided into 10 subfamilies, with nine of these subfamilies corresponding to subfamilies found in other teleosts and one being salmon specific. There is also a large expansion in the number of OlfC genes in one subfamily in Atlantic salmon. Subfamily gene expansions have been identified in other teleosts, and these differences in gene number reflect species-specific evolutionary requirements for olfaction. Total RNA was isolated from the olfactory epithelium and other tissues from a presmolt to examine the expression of the odorant genes. Several of the putative OlfC genes that we identified are expressed only in the olfactory epithelium, consistent with these genes encoding odorant receptors.

Genomic organization of Atlantic salmon (Salmo salar) fatty acid binding protein (fabp2) genes reveals independent loss of duplicate loci in teleosts.

Gene and genome duplications are considered to be driving forces of evolution. The relatively recent genome duplication in the common ancestor of salmonids makes this group of fish an excellent system for studying the re-diploidization process and the fates of duplicate genes. We characterized the structure and genome organization of the intestinal fatty acid binding protein (fabp2) genes in Atlantic salmon as a means of understanding the evolutionary fates of members of this protein family in teleosts. A survey of EST databases identified three unique salmonid fabp2 transcripts (fabp2aI, fabp2aII and fabp2b) compared to one transcript in zebrafish. We screened the CHORI-214 Atlantic salmon BAC library and identified BACs containing each of the three fabp2 genes. Physical mapping, genetic mapping and fluorescence in situ hybridization of Atlantic salmon chromosomes revealed that Atlantic salmon fabp2aI, fabp2aII and fabp2b correspond to separate genetic loci that reside on different chromosomes. Comparative genomic analyses indicated that these genes are related to one another by two genome duplications and a gene loss. The first genome duplication occurred in the common ancestor of all teleosts, giving rise to fabp2a and fabp2b, and the second in the common ancestor of salmonids, producing fabp2aI, fabp2aII, fabp2bI and fabp2bII. A subsequent loss of fabp2bI or fabp2bII gave the complement of fabp2 genes seen in Atlantic salmon today. There is also evidence for independent losses of fabp2b genes in zebrafish and tetraodon. Although there is no evidence for partitioning of tissue expression of fabp2 genes (i.e., sub-functionalization) in Atlantic salmon, the pattern of amino acid substitutions in Atlantic salmon and rainbow trout fabp2aI and fabp2aII suggests that neo-functionalization is occurring.

Distribution of ancestral proto-Actinopterygian chromosome arms within the genomes of 4R-derivative salmonid fishes (Rainbow trout and Atlantic salmon).

BACKGROUND: Comparative genomic studies suggest that the modern day assemblage of ray-finned fishes have descended from an ancestral grouping of fishes that possessed 12-13 linkage groups. All jawed vertebrates are postulated to have experienced two whole genome duplications (WGD) in their ancestry (2R duplication). Salmonids have experienced one additional WGD (4R duplication event) compared to most extant teleosts which underwent a further 3R WGD compared to other vertebrates. We describe the organization of the 4R chromosomal segments of the proto-ray-finned fish karyotype in Atlantic salmon and rainbow trout based upon their comparative syntenies with two model species of 3R ray-finned fishes. RESULTS: Evidence is presented for the retention of large whole-arm affinities between the ancestral linkage groups of the ray-finned fishes, and the 50 homeologous chromosomal segments in Atlantic salmon and rainbow trout. In the comparisons between the two salmonid species, there is also evidence for the retention of large whole-arm homeologous affinities that are associated with the retention of duplicated markers. Five of the 7 pairs of chromosomal arm regions expressing the highest level of duplicate gene expression in rainbow trout share homologous synteny to the 5 pairs of homeologs with the greatest duplicate gene expression in Atlantic salmon. These regions are derived from proto-Actinopterygian linkage groups B, C, E, J and K. CONCLUSION: Two chromosome arms in Danio rerio and Oryzias latipes (descendants of the 3R duplication) can, in most instances be related to at least 4 whole or partial chromosomal arms in the salmonid species. Multiple arm assignments in the two salmonid species do not clearly support a 13 proto-linkage group model, and suggest that a 12 proto-linkage group arrangement (i.e., a separate single chromosome duplication and ancestral fusion/fissions/recombination within the putative G/H/I groupings) may have occurred in the more basal soft-rayed fishes. We also found evidence supporting the model that ancestral linkage group M underwent a single chromosome duplication following the 3R duplication. In the salmonids, the M ancestral linkage groups are localized to 5 whole arm, and 3 partial arm regions (i.e., 6 whole arm regions expected). Thus, 3 distinct ancestral linkage groups are postulated to have existed in the G/H and M lineage chromosomes in the ancestor of the salmonids.

Isolation, characterization and comparison of Atlantic and Chinook salmon growth hormone 1 and 2.

BACKGROUND: Growth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processes in salmonids. The GH gene (gh) in salmonids is represented by duplicated, non-allelic isoforms designated as gh1 and gh2. We have isolated and characterized gh-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinook salmon (Salmo salar and Oncorhynchus tshawytscha) in order to further elucidate our understanding of the conservation and regulation of these loci. RESULTS: BACs containing gh1 and gh2 from both Atlantic and Chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genes for skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1, myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientations relative to gh1 and gh2. Viability of each of these genes was examined by PCR. We show that transposon insertions have occurred differently in the promoters of gh, within and between each species. Other differences within the promoters and intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development. CONCLUSION: A core proximal promoter for transcription of both gh1 and gh2 is conserved between the two species of salmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh1 and gh2. Additionally, organization of transposon families into the BACs containing gh1 and for the BACs containing gh2, are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the gh1- and gh2-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook and other Oncorhynchus GH1s are strikingly different in comparison to the other GHs and this change is not apparent in the surrounding non-coding sequences.

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome.

BACKGROUND: With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering approximately 1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. RESULTS: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with approximately 30x coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (approximately 0.09x coverage) were incorporated. The addition of paired end sequencing reads (additional approximately 26x coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (approximately 10.5x coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly. CONCLUSION: These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.

Genomic organization and characterization of two vomeronasal 1 receptor-like genes (ora1 and ora2) in Atlantic salmon Salmo salar.

Olfactory receptors are encoded by three large multigene superfamilies (OR, V1R and V2R) in mammals. Fish do not possess a vomeronasal system; therefore, it has been proposed that their V1R-like genes be classified as olfactory receptors related to class A G protein-coupled receptors (ora). Unlike mammalian genomes, which contain more than a hundred V1R genes, the five species of teleost fish that have been investigated to date appear to have six ora genes (ora1-6) except for pufferfish that have lost ora1. The common ancestor of salmonid fishes is purported to have undergone a whole genome duplication. As salmonids have a life history that requires the use of olfactory cues to navigate back to their natal habitats to spawn, we set out to determine if ora1 or ora2 is duplicated in a representative species, Atlantic salmon (Salmo salar). We used an oligonucleotide probe designed from a conserved sequence of several teleost ora2 genes to screen an Atlantic salmon BAC library (CHORI-214). Hybridization-positive BACs belonged to a single fingerprint contig of the Atlantic salmon physical map. All were also positive for ora2 by PCR. One of these BACs was chosen for further study, and shotgun sequencing of this BAC identified two V1R-like genes, ora1 and ora2, that are in a head-to-head conformation as is seen in some other teleosts. The gene products, ora1 and ora2, are highly conserved among teleosts. We only found evidence for a single ora1-2 locus in the Atlantic salmon genome, which was mapped to linkage group 6. Fluorescent in situ hybridization (FISH) analysis placed ora1-2 on chromosome 12. Conserved synteny was found surrounding the ora1 and ora2 genes in Atlantic salmon, medaka and three-spined stickleback, but not zebrafish.