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Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

ABSTRACT: BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

SUBMITTER: Pardo BG

PROVIDER: S-EPMC2569028 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

Pardo Belén G BG Fernández Carlos C Millán Adrián A Bouza Carmen C Vázquez-López Araceli A Vera Manuel M Alvarez-Dios José A JA Calaza Manuel M Gómez-Tato Antonio A Vázquez María M Cabaleiro Santiago S Magariños Beatriz B Lemos Manuel L ML Leiro José M JM Martínez Paulino P

BMC veterinary research 20080925

<h4>Background</h4>The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathog ...[more]

PMID: 18817567

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Project description:An EST database from immune tissues was used to design the first high density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60mers) were successfully designed for 2716 out of 3482 unique sequences of the database. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida challenged fish at 3 days post-infection. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide and dye bias) factors using one-colour (1C) and two-colour (2C) -labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random effects variance revealed that technical variation was mostly negligible and biological variation represented the main factor, even using pooled samples. One-colour approach performed at least as well as 2C. A relevant proportion of genes turn out to be differentially labelled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defence response were detected at three days post-challenging. An unbalanced hierarchical design was planned to evaluate the sources of variation in microarray analysis. This design enabled us to consider most sources of noise in microarray analysis and evaluate their weight in the variability of the signal using an equilibrate number of replicates at each level and a not too large number of microarrays. According to this hierarchy, the following within slide sources of variation were assessed from top to bottom: biological (B), RNA extraction (E), labeling (L) and hybridization (H). Additionally, we studied variation between slides (S), the bias produced by labeling with different dyes (Cy3 and Cy5) and compared the performances of one-colour (1C) and two-colour (2C) -labeling approaches. A total of 24 microarrays grouped in three 8x15k slides were used for this design. One pool of five individuals was used as control and other two other pools of five individuals each (B1 and B2) were used as biological replicates. Three replicates were used for evaluating the variation associated with RNA extraction (E1, E2, E3), labeling (L1, L2, L3) and hybridization (H1, H2, H3) within slide. Some of these replicates were in turn hybridized in different slides to assess interslide variation. Three 2C hybridizations with dye swapping (6 microarrays) were performed in the same slide for comparison of 1C and 2C approaches and to analyze dye bias. Normalization within each microarray was carried out using the loess method. Normalization using all microarray data was done by performing the Aquantile method implemented in the limma package. To estimate the variation associated with replicates at the different steps of microarray analysis or to different slides we applied the Pearson correlation coefficient using log2 absolute treatment and treatment/control ratio values. Also an ANOVA of random effects was used to split the total variance into its components. For Dye bias evaluation and 1C vs. 2C comparisons we f estimated the proportion of common differentially expressed (DE) genes after Bonferroni correction at P <0.05 and P<0.01 at fold changes (FC) >1.5, 2 and 3. The degree of concordance between the different dyes and between 1C and 2C approaches was estimated as the proportion of common DE genes at each P and FC.

Project description:BackgroundThe turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies.ResultsA consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups.ConclusionsThe new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.

Dataset Information

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

Publications

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

Similar Datasets

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