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Large-scale sequence analyses of Atlantic cod.

The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.

Genomic divergence between the migratory and stationary ecotypes of Atlantic cod

Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co‐occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome‐wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next‐generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single‐nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North‐east Atlantic.

RNA deep sequencing of the Atlantic cod transcriptome

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.

Digital Marine Bioprospecting : Mining New Neurotoxin Drug Candidates from the Transcriptomes of Cold-Water Sea Anemones

Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.

Approaching marine bioprospecting in hexacorals by RNA deep sequencing

RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-generation sequencing platforms have recently been developed for de novo whole transcriptome analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics (sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential identification of all expressed genes in a sample. When combined to high-throughput bioinformatics and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral transcriptomes with the focus on cold-water sea anemones and related organisms.

Sea anemones possess dynamic mitogenome structures

A notable feature of hexacoral mitogenomes is the presence of complex self-catalytic group I introns. We investigated mitogenome structural variations and evolutionary mechanisms in actiniarian sea anemones based on the complete mitogenome sequence of the cold-water sea anemone species Urticina eques, Bolocera tuediae, Hormathia digitata and Metridium senile, and two isolates of the sub-tropical Aiptasia pulchella. Whole genome sequencing at 50 times coverage of B. tuediae and H. digitata indicated low mtDNA copy number of per haploid nuclear genome and presence of rare haplotypes. A group I intron inserted in ND5 was found to host essential mitochondrial protein genes in all species, and an additional truncated copy of ND5 in B. tuediae. A second group I intron (inserted in COI) that contained a homing endonuclease gene (HEG) was present in all mtDNA examined. Different variants of HEGs were observed, and included expressed elements fused in-frame with upstream exons and free-standing HEGs embedded within the intron. A notable hallmark of HEGs was a high extent of overlap with ribozyme structural elements; the U. eques HEG overlapped with the entire intron. We reconstructed the evolutionary history of the COI intron from insertion at unoccupied cognate sites, through HEG degradation, to intron loss. We also identified a novel insertion element in U. eques that contained two expressed protein-coding genes. An evolutionary analysis of the sea anemone mtDNA genes revealed higher substitution rates in the HEG and the insertion sequence as compared to the other loci, indicating relaxed selective pressures in these elements. We conclude that sea anemone mitogenomes are surprisingly dynamic in structure despite the economical organization and low sequence mutation rate.

Mitogenome polymorphism in a single branch sample revealed by SOLiD deep sequencing of the Lophelia pertusa coral genome

We present an initial genomic analysis of the non-symbiotic scleractinian coral Lophelia pertusa, the dominant cold-water reef-building coral species in the North Atlantic Ocean. A significant fraction of the deep sequencing reads was of mitochondrial and microbial origins. SOLiD deep sequencing reads from fragment library experiments of total DNA and PCR amplified mitogenome generated about 21,000 times and 136,000 times coverage, respectively, of the 16,150 bp mitogenome. Five polymorphic sites that include two non-synonymous sites in the NADH dehydrogenase subunit 5 genes were detected in both experiments. This observation is surprising since anthozoans in general exhibit very low mtDNA sequence variation at intraspecific level compared to nuclear sequences. More than fifty bacterial species associated with the coral isolate were also sequence detected, representing at least ten complete genomes. Most reads, however, were predicted to originate from the Lophelia nuclear genome.

Mitogenome sequence variation in migratory and stationary ecotypes of North-east Atlantic cod.

Sequencing of mitochondrial gene fragments from specimens representing a wide range of geographical locations has indicated limited population structuring in Atlantic cod (Gadus morhua). We recently performed whole genome analysis based on next-generation sequencing of two pooled ecotype samples representing offshore migratory and inshore stationary cod from the North-east Atlantic Ocean. Here we report molecular features and variability of the 16.7kb mitogenome component that was collected from the datasets. These sequences represented more than 25 times coverage of each individual and more than 1100 times coverage of each ecotype sample. We estimated the mitogenome to have evolved 14 times more rapidly than the nuclear genome. Among the 365 single nucleotide polymorphism (SNP) sites identified, 121 were shared between ecotypes, and 151 and 93 were private within the migratory and stationary cod, respectively. We found 323 SNPs to be located in protein coding genes, of which 29 were non-synonymous. One synonymous site in ND2 was likely to be under positive selection. FST measurements indicated weak differentiation in ND1 and ND2 between ecotypes. We conclude that the Atlantic cod mitogenome and the nuclear genome apparently evolved by distinct evolutionary constraints, and that the reproductive isolation observed from whole genome analysis was not visible in the mtDNA sequences.

Mitochondrial genome variation of Atlantic cod

ObjectiveThe objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes.ResultsWe determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species’ distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.

A mitochondrial long noncoding RNA in atlantic cod harbors complex heteroplasmic tandem repeat motifs

Abstract A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2–5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.