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Antarctic fish belonging to Notothenioidei represent an extraordinary example of radiation in the cold. In addition to the absence of hemoglobin, icefish show a number of other striking peculiarities including large-diameter blood vessels, high vascular densities, mitochondria-rich muscle cells, and unusual mitochondrial architecture. In order to investigate the bases of icefish adaptation to the extreme Southern Ocean conditions we sequenced the complete genome of the icefish Chionodraco myersi . Comparative analyses of the icefish genome with those of other teleost species, including two additional white-blooded and five red-blooded notothenioids, provided a new perspective on the evolutionary loss of globin genes. Muscle transcriptome comparative analyses against red-blooded notothenioids as well as temperate fish revealed the peculiar regulation of genes involved in mitochondrial function in icefish. Gene duplication and promoter sequence divergence were identified as genome-wide patterns that likely contributed to the broad transcriptional program underlying the unique features of icefish mitochondria., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the "nervous system development" as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity.

Background: The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam., Results: A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring., Conclusions: The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry. [ABSTRACT FROM AUTHOR]

The aim of the present study was to identify transcriptional modifications and regulatory networks accounting for physiological and metabolic responses to specific nutrients in the liver of young Belgian Blue × Holstein bulls using RNA-sequencing. A larger trial has been carried out in which animals were fed with different diets: 1] a conventional diet; 2] a low-protein/low-mineral diet (low-impact diet) and 3] a diet enriched in n-3 fatty acids (FAs), conjugated linoleic acid (CLA) and vitamin E (nutraceutical diet). The initial hypothesis was that the administration of low-impact and nutraceutical diets might influence the transcriptional profiles in bovine liver and the resultant nutrient fluxes, which are essential for optimal liver function and nutrient interconversion. Results showed that the nutraceutical diet significantly reduced subcutaneous fat covering in vivo and liver pH. Dietary treatments did not affect overall liver fat content, but significantly modified the liver profile of 33 FA traits (out of the total 89 identified by gas-chromatography). In bulls fed nutraceutical diet, the percentage of n-3 and CLA FAs increased around 2.5-fold compared with the other diets, whereas the ratio of n6/n3 decreased 2.5-fold. Liver transcriptomic analyses revealed a total of 198 differentially expressed genes (DEGs) when comparing low-impact, nutraceutical and conventional diets, with the nutraceutical diet showing the greatest effects on liver transcriptome. Functional analyses using ClueGo and Ingenuity Pathway Analysis evidenced that DEGs in bovine liver were variously involved in energy reserve metabolic process, glutathione metabolism, and carbohydrate and lipid metabolism. Modifications in feeding strategies affected key transcription factors regulating the expression of several genes involved in fatty acid metabolism, e.g. insulin-induced gene 1, insulin receptor substrate 2, and RAR-related orphan receptor C. This study provides noteworthy insights into the molecular changes occurring as a result of nutrient variation in diets (aimed at reducing the environmental impact and improving human health) and broadens our understanding of the relationship between nutrients variation and phenotypic effects. [ABSTRACT FROM AUTHOR]

The production of Venerupis decussata relies on wild seed collection, which has been recently compromised due to recruitment failure and severe mortalities. To address this issue and provide an alternative source of seed, artificial spawning and larval rearing programs were developed. However, hatchery-based seed production is a relatively new industry and it is still underdeveloped. A major hurdle in the European clam seed production is the control of spawning and reproduction, which is further hindered by the impossibility of obtaining fertile gametes by gonadal “stripping”, as meiosis re-initiation is constrained to a maturation process along the genital ducts. In the present study, oocytes were collected from 15 females and microarray analyses was performed to investigate gene expression profiles characterizing released and stripped ovarian oocytes. A total of 198 differentially expressed transcripts between stripped and spawned oocytes were detected. Functional analysis carried out on these transcripts highlighted the importance of a few biological processes, which are most probably implicated in the control of oocyte competence. Significant differences were observed for transcripts encoding proteins involved in meiosis progression (e.g. dual specificity phosphatase CDC25), WNT signalling (e.g. frizzled class receptor 8, wingless-type MMTV integration site family member 4), steroid synthesis (e.g. progestin and adipoQ receptor family member 3, cytochrome P450-C17), mRNA processing (e.g. zinc finger protein XlCOF28), calcium regulation (e.g. regucalcin, calmodulin) and ceramide metabolism (ceramidase B, sphingomyelinase). This study provides new information on transcriptional profiles putatively associated with ovarian egg infertility, and suggests potential mechanisms regulating early oocyte development in clams. Genes which were differentially expressed between stripped and spawned oocytes might have a pivotal role during maturation process in the gonadal duct and could be interesting targets for further functional studies aiming to make ovarian oocytes fertilizable. [ABSTRACT FROM AUTHOR]

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs (17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 (82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 (p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation (p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy. [ABSTRACT FROM AUTHOR]

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced. [ABSTRACT FROM AUTHOR]

Antarctic notothenioids radiated over millions of years in subzero waters, evolving peculiar features, such as antifreeze glycoproteins and absence of heat shock response. Icefish, family Channichthyidae, also lack oxygen-binding proteins and display extreme modifications, including high mitochondrial densities in aerobic tissues. A genomic expansion accompanying the evolution of these fish was reported, but paucity of genomic information limits the understanding of notothenioid cold adaptation. We reconstructed and annotated the first skeletal muscle transcriptome of the icefish Chionodraco hamatus providing a new resource for icefish genomics (http://compgen.bio.unipd.it/chamatusbase/, last accessed December 12, 2012). We exploited deep sequencing of this energy-dependent tissue to test the hypothesis of selective duplication of genes involved in mitochondrial function. We developed a bioinformatic approach to univocally assign C. hamatus transcripts to orthology groups extracted from phylogenetic trees of five model species. Chionodraco hamatus duplicates were recorded for each orthology group allowing the identification of duplicated genes specific to the icefish lineage. Significantly more duplicates were found in the icefish when transcriptome data were compared with whole-genome data of model species. Indeed, duplicated genes were significantly enriched in proteins with mitochondrial localization, involved in mitochondrial function and biogenesis. In cold conditions and without oxygen-carrying proteins, energy production is challenging. The combination of high mitochondrial densities and the maintenance of duplicated genes involved in mitochondrial biogenesis and aerobic respiration might confer a selective advantage by improving oxygen diffusion and energy supply to aerobic tissues. Our results provide new insights into the genomic basis of icefish cold adaptation. [ABSTRACT FROM AUTHOR]

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non- model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar- coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild. [ABSTRACT FROM AUTHOR]

Abstract: This study assessed the relationship between the occurrence and function of intronless or single exon genes (SEG) in the genome of five teleost species and their phylogenetic distance. The results revealed that Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculeatus and Danio rerio genomes are respectively comprised of 2.83%, 3.42%, 4.49%, 4.35% and 4.02% SEGs. These SEGs encode for a variety of family proteins including claudins, olfactory receptors and histones that are essential for various biological functions. Subsequently, we predicted and annotated SEGs in three European sea bass, Dicentrarchus labrax chromosomes that we have sequenced, and compared results with those of stickleback (G. aculeatus) homologous chromosomes. While the annotation features of three D. labrax chromosomes revealed 78 (5.30%) intronless genes, comparisons with G. aculeatus showed that SEG composition and their order varied significantly among corresponding chromosomes, even for those with nearly complete synteny. More than half of SEGs identified in most of the species have at least one ortholog multiple exon gene in the same genome, which provides insight to their possible origin by retrotransposition. In spite of the fact that they belong to the same lineage, the fraction of predicted SEGs varied significantly between the genomes analyzed, and only a low fraction of proteins (4.1%) is conserved between all five species. Furthermore, the inter-specific distribution of SEGs as well as the functional categories shared by species did not reflect their phylogenetic relationships. These results indicate that new SEGs are continuously and independently generated after species divergence over evolutionary time as evidenced by the phylogenetic results of single exon claudins genes. Although the origin of SEGs cannot be inferred directly from the phylogeny, our results provide strong support for the idea that retrotransposition followed by tandem duplications is the most probable event that can explain the expansion of SEGs in eukaryotic organisms. [Copyright &y& Elsevier]

Background: Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae. Results: An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon. Conclusion: The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream. [ABSTRACT FROM AUTHOR]

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced. [ABSTRACT FROM AUTHOR]