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.

Ribosomal genes and heat shock proteins as putative markers for chronic, sublethal heat stress in Arctic charr: applications for aquaculture and wild fish.

Arctic charr thrive at high densities and can live in freshwater year round, making this species especially suitable for inland, closed containment aquaculture. However, it is a cold-water salmonid, which both limits where the species can be farmed and places wild populations at particular risk to climate change. Previously, we identified genes associated with tolerance and intolerance to acute, lethal temperature stress in Arctic charr. However, there remained a need to examine the genes involved in the stress response to more realistic temperatures that could be experienced during a summer heat wave in grow-out tanks that are not artificially cooled, or under natural conditions. Here, we exposed Arctic charr to sublethal heat stress of 15-18°C for 72 h, and gill tissues extracted before, during (i.e., at 72 h), immediately after cooling and after 72 h of recovery at ambient temperature (6°C) were used for gene expression profiling by microarray and qPCR analyses. The results revealed an expected pattern for heat shock protein expression, which was highest during heat exposure, with significantly reduced expression (approaching control levels) quickly thereafter. We also found that the expression of numerous ribosomal proteins was significantly elevated immediately and 72 h after cooling, suggesting that the gill tissues were undergoing ribosome biogenesis while recovering from damage caused by heat stress. We suggest that these are candidate gene targets for the future development of genetic markers for broodstock development or for monitoring temperature stress and recovery in wild or cultured conditions.

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.

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.