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.

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Sequencing the genome of the Atlantic salmon (Salmo salar)

Infectious pancreatic necrosis (IPN) is one of the most prevalent and economically devastating diseases in Atlantic salmon (Salmo salar) farming worldwide. The disease causes large mortalities at both the fry- and post-smolt stages. Family selection for increased IPN resistance is performed through the use of controlled challenge tests, where survival rates of sib-groups are recorded. However, since challenge-tested animals cannot be used as breeding candidates, within-family selection is not performed and only half of the genetic variation for IPN resistance is being exploited. DNA markers linked to quantitative trait loci (QTL) affecting IPN resistance would therefore be a powerful selection tool. The aim of this study was to identify and fine-map QTL for IPN-resistance in Atlantic salmon, for use in marker-assisted selection to increase the rate of genetic improvement for this trait. A genome scan was carried out using 10 large full-sib families of challenge-tested Atlantic salmon post-smolts and microsatellite markers distributed across the genome. One major QTL for IPN-resistance was detected, explaining 29% and 83% of the phenotypic and genetic variances, respectively. This QTL mapped to the same location as a QTL recently detected in a Scottish Atlantic salmon population. The QTL was found to be segregating in 10 out of 20 mapping parents, and subsequent fine-mapping with additional markers narrowed the QTL peak to a 4 cM region on linkage group 21. Challenge-tested fry were used to show that the QTL had the same effect on fry as on post-smolt, with the confidence interval for QTL position in fry overlapping the confidence interval found in post-smolts. A total of 178 parents were tested for segregation of the QTL, identifying 72 QTL-heterozygous parents. Genotypes at QTL-heterozygous parents were used to determine linkage phases between alleles at the underlying DNA polymorphism and alleles at single markers or multi-marker haplotypes. One four-marker haplotype was found to be the best predictor of QTL alleles, and was successfully used to deduce genotypes of the underlying polymorphism in 72% of the parents of the next generation within a breeding nucleus. A highly significant population-level correlation was found between deduced alleles at the underlying polymorphism and survival of offspring groups in the fry challenge test, parents with the three deduced genotypes (QQ, Qq, qq) having mean offspring mortality rates of 0.13, 0.32, and 0.49, respectively. The frequency of the high-resistance allele (Q) in the population was estimated to be 0.30. Apart from this major QTL, one other experiment-wise significant QTL for IPN-resistance was detected, located on linkage group 4. The QTL confirmed in this study represents a case of a major gene explaining the bulk of genetic variation for a presumed complex trait. QTL genotypes were deduced within most parents of the 2005 generation of a major breeding company, providing a solid framework for linkage-based MAS within the whole population in subsequent generations. Since haplotype-trait associations valid at the population level were found, there is also a potential for MAS based on linkage disequilibrium (LD). However, in order to use MAS across many generations without reassessment of linkage phases between markers and the underlying polymorphism, the QTL needs to be positioned with even greater accuracy. This will require higher marker densities than are currently available.

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Confirmation and fine-mapping of a major QTL for resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar): population-level associations between markers and trait

The Atlantic salmon genome is in the process of returning to a diploid state after undergoing a whole genome duplication (WGD) event between 25 and100 million years ago. Existing data on the proportion of paralogous sequence variants (PSVs), multisite variants (MSVs) and other types of complex sequence variation suggest that the rediplodization phase is far from over. The aims of this study were to construct a high density linkage map for Atlantic salmon, to characterize the extent of rediploidization and to improve our understanding of genetic differences between sexes in this species. A linkage map for Atlantic salmon comprising 29 chromosomes and 5650 single nucleotide polymorphisms (SNPs) was constructed using genotyping data from 3297 fish belonging to 143 families. Of these, 2696 SNPs were generated from ESTs or other gene associated sequences. Homeologous chromosomal regions were identified through the mapping of duplicated SNPs and through the investigation of syntenic relationships between Atlantic salmon and the reference genome sequence of the threespine stickleback (Gasterosteus aculeatus). The sex-specific linkage maps spanned a total of 2402.3 cM in females and 1746.2 cM in males, highlighting a difference in sex specific recombination rate (1.38:1) which is much lower than previously reported in Atlantic salmon. The sexes, however, displayed striking differences in the distribution of recombination sites within linkage groups, with males showing recombination strongly localized to telomeres. The map presented here represents a valuable resource for addressing important questions of interest to evolution (the process of re-diploidization), aquaculture and salmonid life history biology and not least as a resource to aid the assembly of the forthcoming Atlantic salmon reference genome sequence.

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A dense SNP-based linkage map for Atlantic salmon ( Salmo salar ) reveals extended chromosome homeologies and striking differences in sex-specific recombination patterns

Dense single nucleotide polymorphism (SNP) genotyping arrays provide extensive information on polymorphic variation across the genome of species of interest. Such information can be used in studies of the genetic architecture of quantitative traits and to improve the accuracy of selection in breeding programs. In Atlantic salmon (Salmo salar), these goals are currently hampered by the lack of a high-density SNP genotyping platform. Therefore, the aim of the study was to develop and test a dense Atlantic salmon SNP array. SNP discovery was performed using extensive deep sequencing of Reduced Representation (RR-Seq), Restriction site-Associated DNA (RAD-Seq) and mRNA (RNA-Seq) libraries derived from farmed and wild Atlantic salmon samples (n = 283) resulting in the discovery of > 400 K putative SNPs. An Affymetrix Axiom® myDesign Custom Array was created and tested on samples of animals of wild and farmed origin (n = 96) revealing a total of 132,033 polymorphic SNPs with high call rate, good cluster separation on the array and stable Mendelian inheritance in our sample. At least 38% of these SNPs are from transcribed genomic regions and therefore more likely to include functional variants. Linkage analysis utilising the lack of male recombination in salmonids allowed the mapping of 40,214 SNPs distributed across all 29 pairs of chromosomes, highlighting the extensive genome-wide coverage of the SNPs. An identity-by-state clustering analysis revealed that the array can clearly distinguish between fish of different origins, within and between farmed and wild populations. Finally, Y-chromosome-specific probes included on the array provide an accurate molecular genetic test for sex. This manuscript describes the first high-density SNP genotyping array for Atlantic salmon. This array will be publicly available and is likely to be used as a platform for high-resolution genetics research into traits of evolutionary and economic importance in salmonids and in aquaculture breeding programs via genomic selection.

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Development and validation of a high density SNP genotyping array for Atlantic salmon (Salmo salar)

Next-generation sequencing allows the discovery of large numbers of single nucleotide polymorphisms (SNPs) in species where little genomic information was previously available. Here, we assembled, de novo, over 130 mb of non-normalized cDNA using 454 pyrosequencing data from dwarf and normal lake whitefish and backcross hybrids. Our main goals were to gather a large data set of SNP markers, document their distribution within coding regions, evaluate the effect of species divergence on allele frequencies and combine results with previous genomic studies to identify candidate genes underlying the adaptive divergence of lake whitefish. We identified 6094 putative SNPs in 2674 contigs (mean size: 576 bp, range: 101–6116) and 1540 synonymous and 1734 non-synonymous mutations for a genome-wide non-synonymous to synonymous substitution rate ratio (pN/pS) of 0.37. As expected based on the young age (<15 000 years) of whitefish species pair, the overall level of divergence between them was relatively weak. Yet, 89 SNPs showed pronounced allele frequency differences between sympatric normal and dwarf whitefish. Among these, SNPs in genes annotated to energy metabolic functions were the most abundant and this, in addition to previous experimental data at the gene expression and phenotypic level, brings compelling evidence that genes involved in energy metabolism are prime candidates explaining the adaptive divergence of lake whitefish species pairs. Finally, we unexpectedly identified 44 contigs annotated to transposable elements and these were predominantly composed of backcross hybrids sequences. This indicates an elevated activity of transposable elements, which could potentially contribute to the reduced fitness of hybrids previously documented.

Mining transcriptome sequences towards identifying adaptive single nucleotide polymorphisms in lake whitefish species pairs (Coregonus spp. Salmonidae).

The Atlantic salmon is a species of commercial and ecological significance. Like other salmonids, the species displays residual tetrasomy and a large difference in recombination rate between sexes. Linkage maps with full genome coverage, containing both type I and type II markers, are needed for progress in genomics. Furthermore, it is important to estimate levels of linkage disequilibrium (LD) in the species. In this study, we developed several hundred single nucleotide polymorphism (SNP) markers for the Atlantic salmon, and constructed male and female linkage maps containing SNP and microsatellite markers. We also investigated further the distribution of male and female recombination events across the genome, and estimated levels of LD between pairs of markers. The male map had 29 linkage groups and was 390 cM long. The female map had 30 linkage groups as was 1983 cM long. In total, the maps contained 138 microsatellite markers and 304 SNPs located within genes, most of which were successfully annotated. The ratio of male to female recombination events was either close to zero or very large, indicating that there is little overlap between regions in which male and female crossovers occur. The female map is likely to have close to full genome coverage, while the majority of male linkage groups probably lack markers in telomeric regions where male recombination events occur. Levels of r2 increased with decreasing inter-marker distance in a bimodal fashion; increasing slowly from ~60 cM, and more rapidly more from ~12 cM. Long-ranging LD may be consequence of recent admixture in the population, the population being a 'synthetic' breeding population with contributions from several distinct rivers. Levels of r2 dropped to half its maximum value (above baseline) within 15 cM, and were higher than 0.2 above baseline for unlinked markers ('useful LD') at inter-marker distances less than 5 cM. The linkage map presented here is an important resource for genetic, comparative, and physical mapping of the Atlantic salmon. The female map is likely to have a map coverage that is not far from complete, whereas the male map length is likely to be significantly shorter than the true map, due to suboptimal marker coverage in the apparently small physical regions where male crossovers occur. 'Useful LD' was found at inter-marker distances less than 5 cM.

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Sissel Kjøglum

Papers

A linkage map of the Atlantic salmon (Salmo salar) based on EST-derived SNP markers