Radiation Hybrid Research Articles

Molecular characterization of porcine SARM1 and its role in regulating TLRs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo

Toll-like receptors (TLRs) are important pattern-recognition receptors (PRRs) that trigger innate immune response and mediate acquired immunity. Evidence has shown that SARM1 (sterile-α and TIR motif containing protein 1) is one of five TIR domain-containing adaptor proteins involved in TLRs signaling transduction. In the present study, a full-length cDNA sequence was cloned for the porcine SARM1 gene, which contains nine exons. Using the radiation hybrid mapping approach, we assigned the porcine gene to SSC12 q13. Under the normal condition, porcine SARM1 was highly expressed in brain and spleen. Polyinosinic-polycytidylic acid (poly (I:C)) weakly induced the porcine SARM1 expression in the early stimulation. We found that porcine SARM1 protein is localized in mitochondria and attenuates NF-κB activation induced by stimulation and infection. The quantitative real-time PCR (Q-PCR) analysis showed that the expression of porcine SARM1 significantly decreased in several tissues of Tongcheng pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Gene-interaction network analysis for porcine SARM1 in porcine alveolar macrophages (PAMs) showed that down-regulation of SARM1 gene in infected Tongcheng pig may modulate TRIF-depend TLRs signaling and regulate the expression of disease-resistant genes and inflammatory genes. Our findings provide evidence that porcine SARM1 may play an important role in immune regulation with PRRSV infection.

Molecular characterization of the porcine MTPAP gene associated with meat quality traits: chromosome localization, expression distribution, and transcriptional regulation

MTPAP (mitochondrial poly(A) polymerase) gene plays a role in stabilizing the level of mitochondrial mRNAs and controlling the poly(A) length of human mitochondrial mRNAs. In this study, 2,296 bp partial cDNA sequences of the porcine MTPAP gene were obtained, which contained 1,746 bp full-length coding regions flanked by a 500 bp partial 3′-UTR. The porcine MTPAP gene was assigned to SSC10q14-q16 using the radiation hybrid (IMpRH) panel and chromosome electric location methods. Q-PCR analysis showed that MTPAP was expressed in all analyzed tissues, and has higher expression in heart, liver, skeletal muscles, and fat. One single nucleotide polymorphism g.2421T>A in intron5 of MTPAP gene was identified and detected by DdeI PCR–RFLP. Association of the genotypes with economic traits showed that different genotypes were significantly associated with juiciness, individuals with genotype AT displayed a significantly higher juiciness compared to genotype TT. The C/EBPβ transcription factors was up-regulation the expression of MTPAP by analyzing a series of MTPAP promoter reporter constructs using the dual-luciferase assay system, it indicated that MTPAP gene maybe play a critical role in fat deposition regulation which is regulated by C/EBPβ transcription factor. These findings provide an important basis for further understanding of porcine MTPAP regulation and function in swine.

High-resolution autosomal radiation hybrid maps of the pig genome and their contribution to the genome sequence assembly

BackgroundThe release of the porcine genome sequence offers great perspectives for Pig genetics and genomics, and more generally will contribute to the understanding of mammalian genome biology and evolution. The process of producing a complete genome sequence of high quality, while facilitated by high-throughput sequencing technologies, remains a difficult task. The porcine genome was sequenced using a combination of a hierarchical shotgun strategy and data generated with whole genome shotgun. In addition to the BAC contig map used for the clone-by-clone approach, genomic mapping resources for the pig include two radiation hybrid (RH) panels at two different resolutions. These two panels have been used extensively for the physical mapping of pig genes and markers prior to the availability of the pig genome sequence.ResultsIn order to contribute to the assembly of the pig genome, we genotyped the two radiation hybrid (RH) panels with a SNP array (the Illumina porcineSNP60 array) and produced high density physical RH maps for each pig autosome. We first present the methods developed to obtain high density RH maps with 38,379 SNPs from the SNP array genotyping. We then show how they were useful to identify problems in a draft of the pig genome assembly, and how the RH maps enabled the problems to be corrected in the porcine genome sequence. Finally, we used the RH maps to predict the position of 2,703 SNPs and 1,328 scaffolds currently unplaced on the porcine genome assembly.ConclusionsA complete process, from genotyping of a high density SNP array on RH panels, to the construction of genome-wide high density RH maps, and finally their exploitation for validating and improving a genome assembly is presented here. The study includes the cross-validation of RH based findings with independent information from genetic data and comparative mapping with the Human genome. Several additional resources are also provided, in particular the predicted genomic location of currently unplaced SNPs and associated scaffolds summing up to a total of 72 megabases, that can be useful for the exploitation of the pig genome assembly.

Effects of genome-wide copy number variation on expression in mammalian cells

BackgroundThere is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs).ResultsUnexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements.ConclusionRadiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.

The Complexity of Finding Multiple Solutions to Betweenness and Quartet Compatibility

We show that two important problems that have applications in computational biology are ASP-complete, which implies that, given a solution to a problem, it is NP-complete to decide if another solution exists. We show first that a variation of BETWEENNESS, which is the underlying problem of questions related to radiation hybrid mapping, is ASP-complete. Subsequently, we use that result to show that QUARTET COMPATIBILITY, a fundamental problem in phylogenetics that asks whether a set of quartets can be represented by a parent tree, is also ASP-complete. The latter result shows that Steel’s QUARTET CHALLENGE, which asks whether a solution to QUARTET COMPATIBILITY is unique, is coNP-complete.

Characterization of the porcine FBX07 gene: the first step towards generation of a pig model for Parkinsonian pyramidal syndrome

Parkinsonian pyramidal syndrome, also named pallido-pyramidal syndrome (PKPS), is the combination of early-onset progressive Parkinsonism with pyramidal tract signs. FBXO7, an F-box protein, is a component of modular E3 ubiquitin protein ligases called SCFs (SKP1, cullin, F-box proteins), which functions in phosphorylation-dependent ubiquitination. FBXO7 mutations cause autosomal recessive, early-onset PKPS. Here we report the molecular cloning and characterization of two isoforms of FBXO7 cDNA from pigs. The encoded FBXO7 protein displays a very high homology to human FBXO7 with an amino acid identity of 90%. Phylogenetic analysis demonstrated that porcine FBXO7 is closely related to other mammalian FBXO7 proteins. Furthermore, the genomic structure of the porcine FBXO7 gene was determined. The intron-exon structure is similar to that of the human FBXO7 gene. The promoter sequence for the porcine FBXO7 gene was also identified. A recognition site for miR-301a was found in the 3'UTR region of porcine FBXO7. Investigating the genetic variation in the porcine FBXO7 gene revealed a missense A/G SNP in exon 5. The A/G SNP results in a substitution of an asparagine to a serine residue (N269S). Using a radiation hybrid map the FBXO7 gene was mapped to pig chromosome 5. Real-time quantitative RT-PCR analysis revealed that FBXO7 mRNA is differentially expressed in many tissues and organs, and that FBXO7 transcript can be detected early in embryo development.

Expression profiling analyses of porcine MuRF1 gene and its association with muscle production traits

Muscle specific RING finger protein-1 (MuRF1) is a member of the muscle specific RING finger protein family, and it is specifically expressed in cardiac and skeletal muscle tissues and is involved in myocyte differentiation, development and morphogenesis. In this study the complete open reading frame (ORF) of the porcine MuRF1 gene consisting of 354 amino acids was obtained and it shared 93% and 90% identity with those of the human and mice, respectively. Using the INRA radiation hybrid panel (IMpRH) technique, the MuRF1 gene was assigned to SSC6q21-26, closely linked to microsatellite markers SW1823 and SW709. The tissue distribution patterns revealed that MuRF1 mRNA was exclusively expressed in cardiac and skeletal muscle tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) results displayed that MuRF1 mRNA was up-regulated in Landrace pigs during the prenatal skeletal muscle development stages. A synonymous T/C single nucleotide polymorphism (SNP) was identified in MuRF1 exon 3 and then a Hin6I PCR - RFLP was developed for SNP genotyping in two pig populations. Association of the genotypes with growth and carcass traits showed that different genotypes of MuRF1 were genetically significantly associated with average daily gain from birth to 90 kg and loin muscle area in one experimental population. The study suggested that the porcine MuRF1 gene might affect muscle growth and development, and could be a potential candidate gene for muscle production traits in the pig. Keywords: Molecular cloning, expression profile, mapping, association analysis

Effects of SNPs from the differentially expressed swine odorant binding protein gene on average daily gain

The objective of this study was to analyse genetic mutations in the swine odorant binding protein (OBP), map them on the swine genome, and to investigate the association between genetic variants and growth traits. The tissue sample of intramuscular fat from 10 individuals for each growth stage (0, 90, 150, and 210 days) was used to analyse expression levels of OBP gene, resulting in the highest expression levels at day 90. Sequencing analysis of 307 purebred Yorkshire pigs revealed eight single nucleotide polymorphisms, and the sequence was submitted into the GenBank with an accession number (FJ222446). The SNPs at positions 333 and 508 located in an intron region accounted for significant variation of average daily gain. The analysis of radiation hybrid mapping confirmed a location of the swine OBP gene on chromosome 7 between markers S0064 (475.6 cM) and S0025 (497.4 cM) based on the Sus scrofa radiation hybrid and United States Department of Agriculture swine maps. It is concluded that the identified single nucleotide polymorphism may be used as a useful genetic marker associated with average daily gain.

Mapping, CDNA Cloning and Tissue Expression of the Porcine Thyrotropin-Releasing Hormone Receptor Gene

Thyrotropin-releasing hormone receptor (TRHR) is a G-protein-coupled receptor that plays a crucial role in regulating the hypothalamic-pituitary-thyroid axis by conveying the action of the hypothalamic tripeptide TRH, which is the primary central activator of this hormonal cascade. In the present study, the porcine TRHR (pTRHR) gene was localized to chromosome 4 by Radiation hybrid mapping. Quantitative trait loci affecting average backfat thickness, daily gain, and carcass and meat quality traits have been mapped to the region containing this gene. Further, the full-length cDNA of pTRHR was cloned and sequenced. pTRHR contains an open reading frame encoding 398 amino acids and shares 96.2% amino acid identity to human TRHR. Real-time quantitative RT-PCR showed that the mRNA of pTRHR is expressed in a variety of tissues, with high expression in the brain, hypothalamus, pituitary, testis, and fat tissue. The considerable expression level of TRHR mRNA found in fat tissue indicates potential direct action of TRH on lipocyte might exist. Additionally, two alternative spliced transcript variants of pTRHR were also isolated in this study. Our data provided basic molecular information which will be useful for further investigation on pTRHR gene.

Identification of alternatively spliced Dab1 and Fyn isoforms in pig

BackgroundDisabled-1 (Dab1) is an adaptor protein that is essential for the intracellular transduction of Reelin signaling, which regulates the migration and differentiation of postmitotic neurons during brain development in vertebrates. Dab1 function depends on its tyrosine phosphorylation by Src family kinases, especially Fyn.ResultsWe have isolated alternatively spliced forms of porcine Dab1 from brain (sDab1) and liver (sDab1-Li) and Fyn from brain (sFyn-B) and spleen (sFyn-T). Radiation hybrid mapping localized porcine Dab1 (sDab1) and Fyn (sFyn) to chromosomes 6q31-35 and 1p13, respectively. Real-time quantitative RT-PCR (qRT-PCR) demonstrated that different isoforms of Dab1 and Fyn have tissue-specific expression patterns, and sDab1 and sFyn-B display similar temporal expression characteristics in the developing porcine cerebral cortex and cerebellum. Both sDab1 isoforms function as nucleocytoplasmic shuttling proteins. It was further shown that sFyn phosphorylates sDab1 at tyrosyl residues (Tyr) 185, 198/200 and 232, whereas sDab1-Li was phosphorylated at Tyr 185 and Tyr 197 (corresponding to Y232 in sDab1) in vitro.ConclusionsAlternative splicing generates natural sDab1-Li that only carries Y185 and Y197 (corresponding to Y232 in sDab1) sites, which can be phosphorylated by Fyn in vitro. sDab1-Li is an isoform that is highly expressed in peripheral organs. Both isoforms are suggested to be nucleocytoplasmic shuttling proteins. Our results imply that the short splice form sDab1-Li might regulate cellular responses to different cell signals by acting as a dominant negative form against the full length sDab1 variant and that both isoforms might serve different signaling functions in different tissues.

Molecular characterization of the porcine JHDM1A gene associated with average daily gain: evaluation its role in skeletal muscle development and growth

JHDM1A, a member of the JHDM (JmjC-domain-containing histone demethylase) family, plays an central role in gene silencing, cell cycle, cell growth and cancer development through histone H3K36 demethylation modification. Here reported the cloning, expression, chromosomal location and association analysis with growth traits of porcine JHDM1A gene. Sequence analysis showed that the porcine JHDM1A gene encodes 1,162 amino acids and contains JmjC, F-box, and CXXC zinc-finger domains, which coding sequence and deduced protein shares 91 and 99% similarity with human JHDM1A, respectively. Spatio-Temporal expression analysis indicated that the mRNA expression of porcine JHDM1A had significantly higher levels in the middle (65 days) and later (90 days) period's embryo skeletal muscle than that of 33 days, and showed a ubiquitously expression but with the highest abundance in kidney, lung and liver of an adult pig. Radiation hybrid mapping and the following linkage mapping data indicate that JHDM1A maps to 2p17 region of pig chromosome 2 (SSC2). Allele frequency differences were detected in different pig breeds and an association study was performed with a SNP within 3'UTR. The results showed that there is a tendency for allele frequencies to differ between the fast growth breeds (Yorkshire) and slow growth pig breeds (Qingping pigs, Yushan Black pigs, Erhualian pigs and Dahuabai pigs). The association analysis using a Berkshire × Yorkshire F(2) population indicated that the C224G polymorphism had a highly significant association with average daily gain on test (P < 0.01), a trend association with average back fat thickness (P < 0.07), and significant associations (P < 0.01) on percent of average drip loss, Fiber Type II Ratio, muscle shear force and average lactate content in μmol/g. This study provides the first evidence that JHDM1A is differentially expressed in porcine embryonic skeletal muscle and associated with meat growth and quality traits.

Enriching the bovine microsatellite Radiation Hybrid map with AFLP® markers

Genomic maps of ordered markers and expressed sequences are basic tools for the identification and positional cloning of QTLs and genes involved in economic traits in farm animals. The analysis of donor/hamster Radiation Hybrid (RH) panels represent a powerful method to map genes ESTs and markers. RH maps are generally characterised and anchored to linkage maps using SSR markers employing a quite large experimental effort. In addition, the number of available SSRs may in some case be insufficient to assemble complete RH maps, particularly in high-resolution panels. We have adapted the high-throughput AFLP® technology (Vos et al., 1995) to the typing of RH cell lines, and have used it to increase the number of anchor points in the bovine RH map.

A high-resolution comparative map of porcine chromosome 4 (SSC4)

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Statistical confidence measures for genome maps: application to the validation of genome assemblies

Genome maps are imperative to address the genetic basis of the biology of an organism. While a growing number of genomes are being sequenced providing the ultimate genome maps-this being done at an even faster pace now using new generation sequencers-the process of constructing intermediate maps to build and validate a genome assembly remains an important component for producing complete genome sequences. However, current mapping approach lack statistical confidence measures necessary to identify precisely relevant inconsistencies between a genome map and an assembly. We propose new methods to derive statistical measures of confidence on genome maps using a comparative model for radiation hybrid data. We describe algorithms allowing to (i) sample from a distribution of maps and (ii) exploit this distribution to construct robust maps. We provide an example of application of these methods on a dog dataset that demonstrates the interest of our approach. Methods are implemented in two freely available softwares: Carthagene (http://www.inra.fr/mia/T/CarthaGene/) and a companion software (metamap, available at: http://snp.toulouse.inra.fr/~servin/index.cgi/Metamap).

Integrative mapping analysis of chicken microchromosome 16 organization

BackgroundThe chicken karyotype is composed of 39 chromosome pairs, of which 9 still remain totally absent from the current genome sequence assembly, despite international efforts towards complete coverage. Some others are only very partially sequenced, amongst which microchromosome 16 (GGA16), particularly under-represented, with only 433 kb assembled for a full estimated size of 9 to 11 Mb. Besides the obvious need of full genome coverage with genetic markers for QTL (Quantitative Trait Loci) mapping and major genes identification studies, there is a major interest in the detailed study of this chromosome because it carries the two genetically independent MHC complexes B and Y. In addition, GGA16 carries the ribosomal RNA (rRNA) genes cluster, also known as the NOR (nucleolus organizer region). The purpose of the present study is to construct and present high resolution integrated maps of GGA16 to refine its organization and improve its coverage with genetic markers.ResultsWe developed 79 STS (Sequence Tagged Site) markers to build a physical RH (radiation hybrid) map and 34 genetic markers to extend the genetic map of GGA16. We screened a BAC (Bacterial Artificial Chromosome) library with markers for the MHC-B, MHC-Y and rRNA complexes. Selected clones were used to perform high resolution FISH (Fluorescent In Situ Hybridization) mapping on giant meiotic lampbrush chromosomes, allowing meiotic mapping in addition to the confirmation of the order of the three clusters along the chromosome. A region with high recombination rates and containing PO41 repeated elements separates the two MHC complexes.ConclusionsThe three complementary mapping strategies used refine greatly our knowledge of chicken microchromosome 16 organisation. The characterisation of the recombination hotspots separating the two MHC complexes demonstrates the presence of PO41 repetitive sequences both in tandem and inverted orientation. However, this region still needs to be studied in more detail.

Chromosome mapping of five differently expressed miRNAs in porcine skeletal muscle development (Brief Report)

Abstract. MicroRNAs (miRNAs) are a class of short, non-coding regulatory RNAs, which are approximately 22 nucleotides in length. Typically, miRNAs negatively regulate gene expression by binding with the 3' untranslated region (UTR) of its regulatory target mRNAs. MicroRNAs are known to play diverse roles in fundamental biological processes, such as proliferation, differentiation and apoptosis (Bartel 2004, 2009). It has been reported that miR-1, miR-133, miR-181 and miR-206 play important roles in skeletal muscle proliferation and hypertrophy (Callis et al. 2007, McCarthy -Esser 2007). We have detected porcine miRNA expression profiles during different stage of skeletal muscle development and a total of 140 miRNAs were differentially expressed (HUANG et al. 2008). In this study, we mapped five differentially expressed miRNAs (mir-29c, mir-103-1, mir-127, mir-193b and mir-218-1) using the radiation hybrid (IMpRH) panel (YERLE et al. 1998).

The molecular characterization and associations of porcine cardiomyopathy asssociated 5 (CMYA5) gene with carcass trait and meat quality

The cardiomyopathy associated 5 (CMYA5) gene was also called TRIM76, which was belonged to the tripartite motif super family of proteins (TRIM). It was a direct transcriptional target for MEF2A and it played an important role in myofibrillogenesis. In the present study, a 12056 bp cDNA sequence of the porcine CMYA5 gene was obtained by RT-PCR. The sequence encoded a large protein consisting of 4003 amino acids and the carboxyl terminus of the predicted CMYA5 protein comprised of a B-box coiled-coil, two fibronectin type III (FN3) repeats, and SPRY domains. The porcine CMYA5 gene was assigned to chromosome 2q21-24 by using the radiation hybrid (IMpRH) panel, and it was significantly linked to microsatellite Sw1602 with LOD scores of 6.74. Semi-quantitative RT-PCR revealed that the porcine CMYA5 gene was broadly expressed in all seven tissues(heart, liver, spleen, lung, kidney, skeletal muscle and adipose)harvested from different developmental stages(new born, five weeks and adult tongcheng pigs), with a high level in heart and skeletal muscle. One SNP (A7189C), leading to the amino acid alteration from the Ile residue to the Leu residue, was found and detected by BspTI PCR-restriction fragment length polymorphism. The association analysis revealed that the substitution of A7189C had significant associations with the percentage of ham (p < 0.05), water loss (p < 0.01) and intramuscular fat (p < 0.05). These results provide the evidence that the porcine CMYA5 gene can act as a potential candidate gene affecting pig meat quality.

Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR-RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.

Molecular characterization, chromosomal localization, expression profile and association analysis with carcass traits of the porcine dickkopf homolog1 gene

DKK1 (dickkopf homolog 1) is a potent inhibitor of the canonical Wnt/β-cantenin signalling pathway, which plays a pivotal role in myogenesis, adipogenesis, and many other crucial biological processes. In this study, DKK1 was assigned to porcine 14q25-26 by using the radiation hybrid (IMpRH) panel. A G1757A single nucleotide polymorphism site by Csp6I PCR-RFLP was identified. Association analysis showed that different genotypes were associated with loin muscle area (P = 0.0281). Semi-quantitative-RT-PCR analysis revealed that DKK1 was highly expressed in spleen and lymph node at two developmental stages, while in skeletal muscle, further real-time PCR quantified that DKK1 was down-regulated in Large White pigs compared to Tongcheng pigs, accompanied by the down-regulation of CTTNB1 and TCF4, the up-regulation of LRP6, suggesting that the phenotypic difference between lean and obese pigs might be correlated with the activity of Wnt/β-cantenin signalling pathway.

Gilthead sea bream ( Sparus auratus) and European sea bass ( Dicentrarchus labrax) expressed sequence tags: Characterization, tissue-specific expression and gene markers

The gilthead sea bream, Sparus auratus, and the European sea bass, Dicentrarchus labrax, are two of the most important marine species cultivated in Southern Europe. This study aimed at increasing genomic resources for the two species and produced and annotated two sets of 30,000 expressed sequence tags (EST) each from 14 normalized tissue-specific cDNA libraries from sea bream and sea bass. Clustering and assembly of the ESTs formed 5268 contigs and 12,928 singletons for sea bream and 4573 contigs and 13,143 singletons for sea bass, representing 18,196 and 17,716 putative unigenes, respectively. Assuming a similar number of genes in sea bass, sea bream and in the model fish Gasterosteus aculeatus genomes, it was estimated that approximately two thirds of the sea bream and the sea bass transcriptomes were covered by the unigene collections. BLAST sequence similarity searches (using a cut off of e-value < 10 −5) against fully the curated SwissProt (and TrEMBL) databases produced matches of 28%(37%) and 43%(53%) of the sea bream and sea bass unigene datasets respectively, allowing some putative designation of function. A comparative approach is described using human Ensembl peptide ID homolog's for functional annotation, which increased the number of unigenes with GO terms assigned and resulted in more GO terms assigned per unigene. This allowed the identification of tissue-specific genes using enrichment analysis for GO pathways and protein domains. The comparative annotation approach represents a good strategy for transferring more relevant biological information from highly studied species to genomic resource poorer species. It was possible to confirm by interspecies mRNA-to-genomic alignments 25 and 21 alternative splice events in sea bream and sea bass genes, respectively. Even using normalized cDNA from relatively few pooled individuals it was possible to identify 1145 SNPs and 1748 microsatellites loci for genetic marker development. The EST data are being applied to a range of projects, including the development microarrays, genetic and radiation hybrid maps and QTL genome scans. This highlights the important role of ESTs for generating genetic and genomic resources of aquaculture species.