Abstract
BackgroundAquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on biological processes in farmed species and overcome such bottlenecks. Here we present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture.ResultsA public data base was constructed, consisting of 19,734 unique clusters (3,563 contigs and 16,171 singletons). Functional annotation was obtained for 8,021 clusters. Over 4,000 sequences were also associated with a GO entry. Two 60mer probes were designed for each gene and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Platform reproducibility and accuracy were assessed on two early stages of sea bream development (one-day and four days old larvae). Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. A two class SAM test identified 1,050 differentially expressed genes between the two developmental stages. Functional analysis suggested that down-regulated transcripts (407) in older larvae are mostly essential/housekeeping genes, whereas tissue-specific genes are up-regulated in parallel with the formation of key organs (eye, digestive system). Cross-validation of microarray data was carried out using quantitative qRT-PCR on 11 target genes, selected to reflect the whole range of fold-change and both up-regulated and down-regulated genes. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates. Good concordance between qRT-PCR and microarray data was observed between 2- and 7-fold change, while fold-change compression in the microarray was present for differences greater than 10-fold in the qRT-PCR.ConclusionA highly reliable oligo-microarray platform was developed and validated for the gilthead sea bream despite the presently limited knowledge of the species transcriptome. Because of the flexible design this array will be able to accommodate additional probes as soon as novel unique transcripts are available.
Highlights
Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved
We used all the available expressed sequence tags (ESTs) from the gilthead sea bream to design two longer (60mer) probes for each transcript in-situ synthesized on glass slides using Agilent SurePrintTM technology
Only normalized cDNA libraries were used to produce the vast majority of clustered ESTs, this determined a few number of contigs compared to singletons
Summary
Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. We present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture. The gilthead sea bream (Sparus aurata Linnaeus, 1758) is a marine teleost that belongs to the family Sparidae. Sparids are of great importance for fisheries and aquaculture, being excellent food fish, with high commercial value S. aurata is one of the most prominent, with an average cultured production of 100 million metric tonnes per year. The great importance of the gilthead sea bream for marine aquaculture has fuelled an increasing number of studies in many different areas such as immunology, endocrinology, bone morphology, and muscle physiology. A first generation cDNA microarray was recently reported [1], a radiation hybrid (RH) map has been constructed [2] and further improved with over 1,000 markers [3].
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