miRNAs Predicted to Regulate Host Anti-viral Gene Pathways in IPNV-Challenged Atlantic Salmon Fry Are Affected by Viral Load, and Associated With the Major IPN Resistance QTL Genotypes in Late Infection

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 “high viral load responding” miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes

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

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-rayfinned 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

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr–smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)–salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community

Comparative Genome Analysis of the Primary Sex-Determining Locus in Salmonid Fishes

We compared the Y-chromosome linkage maps for four salmonid species (Arctic charr, Salvelinus alpinus; Atlantic salmon, Salmo salar; brown trout, Salmo trutta; and rainbow trout, Oncorhynchus mykiss) and a putative Y-linked marker from lake trout (Salvelinus namaycush). These species represent the three major genera within the subfamily Salmoninae of the Salmonidae. The data clearly demonstrate that different Y-chromosomes have evolved in each of the species. Arrangements of markers proximal to the sex-determining locus are preserved on homologous, but different, autosomal linkage groups across the four species studied in detail. This indicates that a small region of DNA has been involved in the rearrangement of the sex-determining region. Placement of the sex-determining region appears telomeric in brown trout, Atlantic salmon, and Arctic charr, whereas an intercalary location for SEX may exist in rainbow trout. Three hypotheses are proposed to account for the relocation: translocation of a small chromosome arm; transposition of the sex-determining gene; or differential activation of a primary sex-determining gene region among the species