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Halifax, Canada

Hori T.S.,Memorial University of Newfoundland | Gamperl A.K.,Memorial University of Newfoundland | Afonso L.O.B.,British Columbia Center for Aquatic Health science | Johnson S.C.,Northwest Atlantic Fisheries Center | And 4 more authors.
BMC Genomics

Background: Daily and seasonal changes in temperature are challenges that fish within aquaculture settings cannot completely avoid, and are known to elicit complex organismal and cellular stress responses. We conducted a large-scale gene discovery and transcript expression study in order to better understand the genes that are potentially involved in the physiological and cellular aspects of stress caused by heat-shock. We used suppression subtractive hybridization (SSH) cDNA library construction and characterization to identify transcripts that were dysregulated by heat-shock in liver, skeletal muscle and head kidney of Atlantic cod. These tissues were selected due to their roles in metabolic regulation, locomotion and growth, and immune function, respectively. Fish were exposed for 3 hours to an 8°C elevation in temperature, and then allowed to recover for 24 hours at the original temperature (i.e. 10°C). Tissue samples obtained before heat-shock (BHS), at the cessation of heat-shock (CS), and 3, 12, and 24 hours after the cessation of heat-shock (ACS), were used for reciprocal SSH library construction and quantitative reverse transcription - polymerase chain reaction (QPCR) analysis of gene expression using samples from a group that was transferred but not heat-shocked (CT) as controls.Results: We sequenced and characterized 4394 ESTs (1524 from liver, 1451 from head kidney and 1419 from skeletal muscle) from three "forward subtracted" libraries (enriched for genes up-regulated by heat-shock) and 1586 from the liver "reverse subtracted" library (enriched for genes down-regulated by heat-shock), for a total of 5980 ESTs. Several cDNAs encoding putative chaperones belonging to the heat-shock protein (HSP) family were found in these libraries, and "protein folding" was among the gene ontology (GO) terms with the highest proportion in the libraries. QPCR analysis of HSP90α and HSP70-1 (synonym: HSPA1A) mRNA expression showed significant up-regulation in all three tissues studied. These transcripts were more than 100-fold up-regulated in liver following heat-shock. We also identified HSP47, GRP78 and GRP94-like transcripts, which were significantly up-regulated in all 3 tissues studied. Toll-like receptor 22 (TLR22) transcript, found in the liver reverse SSH library, was shown by QPCR to be significantly down-regulated in the head kidney after heat-shock.Conclusion: Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress. Moreover, the transcript for one key immune response gene (TLR22) was down-regulated by heat-shock, and this down-regulation may be a component of heat-induced immunosuppression. © 2010 Hori et al; licensee BioMed Central Ltd. Source

Garber A.F.,Huntsman Marine Science Center | Garber A.F.,Canadian Department of Fisheries and Oceans | Tosh J.J.,University of Guelph | Fordham S.E.,Huntsman Marine Science Center | And 7 more authors.

Differential survival up to harvest was evaluated for 73 communally reared families of Atlantic cod (Gadus morhua). Progeny from known family crosses were produced and mixed as fertilized eggs (2-3 families per incubator), newly hatched larvae, or as ~. 7. g juveniles to evaluate differences in contribution among families based on stage of mixing. All progeny were stocked into a single sea cage. Progeny were harvested and fin tissue collected at 991-1037. days post-fertilization (n=2001). A multiplex of six microsatellite markers was used to genotype broodstock and progeny for parentage assignment using PROBMAX. Of the progeny assigned (n=1996), only 34 were from 10 of the 27 families mixed as fertilized eggs. In contrast, all families mixed as hatched larvae or as ~. 7. g juveniles were represented (536 progeny from 11 of 11 families; 1426 progeny from 35 of 35 families, respectively). The differences in family survival were affected by sire and stage of mixing. The results indicate progeny should be combined as larvae or juveniles to reduce loss of genetic diversity in communally reared populations. Heritabilities for total body, carcass and fillet weight at time of harvest were ≥ 0.35 and suggest that the developing Canadian Atlantic cod aquaculture industry should benefit from genetic improvement of these traits. However, gonad weight was less heritable (0.11), indicating that sexual maturation will respond less favorably to traditional selective breeding methods. © 2010 Elsevier B.V. Source

Hubert S.,The Atlantic Genome Center | Hubert S.,University of Sainte-Anne | Higgins B.,The Atlantic Genome Center | Borza T.,The Atlantic Genome Center | Bowman S.,The Atlantic Genome Center
BMC Genomics

Background: Atlantic cod (Gadus morhua) is a species with increasing economic significance for the aquaculture industry. The genetic improvement of cod will play a critical role in achieving successful large-scale aquaculture. While many microsatellite markers have been developed in cod, the number of single nucleotide polymorphisms (SNPs) is currently limited. Here we report the identification of SNPs from sequence data generated by a large-scale expressed sequence tag (EST) program, focusing on fish originating from Canadian waters.Results: A total of 97976 ESTs were assembled to generate 13448 contigs. We detected 4753 SNPs that met our selection criteria (depth of coverage ≥ 4 reads; minor allele frequency > 25%). 3072 SNPs were selected for testing. The percentage of successful assays was 75%, with 2291 SNPs amplifying correctly. Of these, 607 (26%) SNPs were monomorphic for all populations tested. In total, 64 (4%) of SNPs are likely to represent duplicated genes or highly similar members of gene families, rather than alternative alleles of the same gene, since they showed a high frequency of heterozygosity. The remaining polymorphic SNPs (1620) were categorised as validated SNPs. The mean minor allele frequency of the validated loci was 0.258 (± 0.141). Of the 1514 contigs from which validated SNPs were selected, 31% have a significant blast hit. For the SNPs predicted to occur in coding regions (141), we determined that 36% (51) are non-synonymous. Many loci (1033 SNPs; 64%) are polymorphic in all populations tested. However a small number of SNPs (184) that are polymorphic in the Western Atlantic were monomorphic in fish tested from three European populations. A preliminary linkage map has been constructed with 23 major linkage groups and 924 mapped SNPs.Conclusions: These SNPs represent powerful tools to accelerate the genetic improvement of cod aquaculture. They have been used to build a genetic linkage map that can be applied to quantitative trait locus (QTL) discovery. Since these SNPs were generated from ESTs, they are linked to specific genes. Genes that map within QTL intervals can be prioritized for testing to determine whether they contribute to observed phenotypes. © 2010 Hubert et al; licensee BioMed Central Ltd. Source

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