miR-430 microRNA Family in Fishes: Molecular Characterization and Evolution.

The miR-430 microRNA family has been described in multiple fish species as one of the first microRNAs expressed by the zygote. It has been suggested that this family is implicated in maternal mRNA elimination, but may also play a role in steroidogenesis, sexual differentiation, and flatfish metamorphosis. The miR-430 sequences have been found in multiple-copy tandem clusters but evidence of their conservation outside of teleost fishes is scarce. In the present study, we have characterized the tandem repeats organization of these microRNAs in different fish species, both model and of interest in aquaculture. A phylogenetic analysis of this family has allowed us to identify that the miR-430 duplication, which took place before the Chondrostei and Neopterygii groups' divergence, has resulted in three variants ("a", "b", and "c"). According to our data, variant "b" is the most closely related to the ancestral sequence. Furthermore, we have detected isolated instances of the miR-430 repeat subunit in some species, which suggests that this microRNA family may be affected by DNA rearrangements. This study provides new data about the abundance, variability, and organization of the miR-430 family in fishes.

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis.

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.

Genomic Characterization of hox Genes in Senegalese Sole (Solea senegalensis, Kaup 1858): Clues to Evolutionary Path in Pleuronectiformes.

The Senegalese sole (Solea senegalensis, Kaup 1858), a marine flatfish, belongs to the Pleuronectiformes order. It is a commercially important species for fisheries and aquaculture. However, in aquaculture, several production bottlenecks have still to be resolved, including skeletal deformities and high mortality during the larval and juvenile phase. The study aims to characterize the hox gene clusters in S. senegalensis to understand better the developmental and metamorphosis process in this species. Using a BAC library, the clones that contain hox genes were isolated, sequenced by NGS and used as BAC-FISH probes. Subsequently the hox clusters were studied by sequence analysis, comparative genomics, and cytogenetic and phylogenetic analysis. Cytogenetic analysis demonstrated the localization of four BAC clones on chromosome pairs 4, 12, 13, and 16 of the Senegalese sole cytogenomic map. Comparative and phylogenetic analysis showed a highly conserved organization in each cluster and different phylogenetic clustering in each hox cluster. Analysis of structural and repetitive sequences revealed accumulations of polymorphisms mediated by repetitive elements in the hoxba cluster, mainly retroelements. Therefore, a possible loss of the hoxb7a gene can be established in the Pleuronectiformes lineage. This work allows the organization and regulation of hox clusters to be understood, and is a good base for further studies of expression patterns.

Integration of Maps Enables a Cytogenomics Analysis of the Complete Karyotype in Solea senegalensis.

The Pleuronectiformes order, which includes several commercially-important species, has undergone extensive chromosome evolution. One of these species is Solea senegalensis, a flatfish with 2n = 42 chromosomes. In this study, a cytogenomics approach and integration with previous maps was applied to characterize the karyotype of the species. Synteny analysis of S. senegalensis was carried out using two flatfish as a reference: Cynoglossus semilaevis and Scophthalmus maximus. Most S. senegalensis chromosomes (or chromosome arms for metacentrics and submetacentrics) showed a one-to-one macrosyntenic pattern with the other two species. In addition, we studied how repetitive sequences could have played a role in the evolution of S. senegalensis bi-armed (3, and 5-9) and acrocentric (11, 12 and 16) chromosomes, which showed the highest rearrangements compared with the reference species. A higher abundance of TEs (Transposable Elements) and other repeated elements was observed adjacent to telomeric regions on chromosomes 3, 7, 9 and 16. However, on chromosome 11, a greater abundance of DNA transposons was detected in interstitial BACs. This chromosome is syntenic with several chromosomes of the other two flatfish species, suggesting rearrangements during its evolution. A similar situation was also found on chromosome 16 (for microsatellites and low complexity sequences), but not for TEs (retroelements and DNA transposons). These differences in the distribution and abundance of repetitive elements in chromosomes that have undergone remodeling processes during the course of evolution also suggest a possible role for simple repeat sequences in rearranged regions.

Cytogenomics Unveil Possible Transposable Elements Driving Rearrangements in Chromosomes 2 and 4 of Solea senegalensis.

Cytogenomics, the integration of cytogenetic and genomic data, has been used here to reconstruct the evolution of chromosomes 2 and 4 of Solea senegalensis. S. senegalensis is a flat fish with a karyotype comprising 2n = 42 chromosomes: 6 metacentric + 4 submetacentric + 8 subtelocentric + 24 telocentric. The Fluorescence in situ Hybridization with Bacterial Artificial Chromosomes (FISH-BAC) technique was applied to locate BACs in these chromosomes (11 and 10 BACs in chromosomes 2 and 4, respectively) and to generate integrated maps. Synteny analysis, taking eight reference fish species (Cynoglossus semilaevis, Scophthalmus maximus, Sparus aurata, Gasterosteus aculeatus, Xiphophorus maculatus, Oryzias latipes, Danio rerio, and Lepisosteus oculatus) for comparison, showed that the BACs of these two chromosomes of S. senegalensis were mainly distributed in two principal chromosomes in the reference species. Transposable Elements (TE) analysis showed significant differences between the two chromosomes, in terms of number of loci per Mb and coverage, and the class of TE (I or II) present. Analysis of TE divergence in chromosomes 2 and 4 compared to their syntenic regions in four reference fish species (C. semilaevis, S. maximus, O. latipes, and D. rerio) revealed differences in their age of activity compared with those species but less notable differences between the two chromosomes. Differences were also observed in peaks of divergence and coverage of TE families for all reference species even in those close to S. senegalensis, like S. maximus and C. semilaevis. Considered together, chromosomes 2 and 4 have evolved by Robertsonian fusions, pericentric inversions, and other chromosomal rearrangements mediated by TEs.

The genomic structure of the highly-conserved dmrt1 gene in Solea senegalensis (Kaup, 1868) shows an unexpected intragenic duplication.

Knowing the factors responsible for sex determination in a species has significant theoretical and practical implications; the dmrt1 gene (Doublesex and Mab-3 (DM)-related Transcription factor 1) plays this role in diverse animal species. Solea senegalensis is a commercially important flat fish in which females grow 30% faster than males. It has 2n = 42 chromosomes and an XX / XY chromosome system for sex determination, without heteromorph chromosomes but with sex proto-chromosome. In the present study, we are providing the genomic structure and nucleotide sequence of dmrt1 gene obtained from cDNA from male and female adult gonads. A cDNA of 2027 containing an open-reading frame (ORF) of 1206 bp and encoding a 402 aa protein it is described for dmrt1 gene of S. senegalensis. Multiple mRNA isoforms indicating a high variable system of alternative splicing in the expression of dmrt1 of the sole in gonads were studied. None isoforms could be related to sex of individuals. The genomic structure of the dmrt1 of S. senegalensis showed a gene of 31400 bp composed of 7 exons and 6 introns. It contains an unexpected duplication of more than 10399 bp, involving part of the exon I, exons II and III and a SINE element found in the sequence that it is proposed as responsible for the duplication. A mature miRNA of 21 bp in length was localized at 336 bp from exon V. Protein-protein interacting networks of the dmrt1 gene showed matches with dmrt1 protein from Cynoglossus semilaevis and a protein interaction network with 11 nodes (dmrt1 plus 10 other proteins). The phylogenetic relationship of the dmrt1 gene in S. senegalensis is consistent with the evolutionary position of its species. The molecular characterization of this gene will enhance its functional analysis and the understanding of sex differentiation in Solea senegalensis and other flatfish.

Gene clusters related to metamorphosis in Solea senegalensis are highly conserved.

The flatfish, Solea senegalensis has considerable scientific interest and commercial value. The metamorphosis in this species occurs between 12 and 19 days after hatching and it takes about 1 week to complete. Eleven Bacterial Artificial Chromosomes (BAC) clones containing the various candidate genes involved in the process of metamorphosis: thyroxine 5 deiodinase 3 (dio3); forkhead box protein E4 (foxe4); melatonin receptor type 1C (mel1c); calsequestrin 1b (casq1b); thyrotropin subunit beta (tshß); thyrotropin-releasing hormone receptor 1, 2, and 3 (trhr1, trhr2, trhr3); thyroid hormone receptor α a and b (thrαa, thrαb); and thyroid hormone receptor beta (thrß) were analyzed by multiple Fluorescence in situ Hybridization (mFISH) and Next Generation Sequencing (NGS) techniques. The mFISH technique localized the 11 BAC clones on 12 different chromosome pairs because three of them, specifically the trhr1a, trhr2 and thrß BAC clones, showed double signals. This signal duplication indicates a duplication of the genomic region inserted within the BAC clone, which provides evidence for the Teleost-Specific Whole Genome Duplication (TS-WGD). Micro-synteny and phylogenetic analysis showed that Cynoglossus semilaevis is the nearest species to S. senegalensis and that Danio rerio is the most distant one. The tshß BAC clone was highly conserved as the genes belonging to this BAC were located on a single chromosome in all the species studied. These genes participate in proliferation, migration and cell-death, which are key processes during metamorphosis. Overall, micro-synteny analysis showed that most candidate genes are found in conserved genomic surroundings.

A Comprehensive Integrated Genetic Map of the Complete Karyotype of Solea senegalensis (Kaup 1858).

Solea senegalensis aquaculture production has experienced a great increase in the last decade and, consequently, the genome knowledge of the species is gaining attention. In this sense, obtaining a high-density genome mapping of the species could offer clues to the aquaculture improvement in those aspects not resolved so far. In the present article, a review and new processed data have allowed to obtain a high-density BAC-based cytogenetic map of S. senegalensis beside the analysis of the sequences of such BAC clones to achieve integrative data. A total of 93 BAC clones were used to localize the chromosome complement of the species and 588 genes were annotated, thus almost reaching the 2.5% of the S. senegalensis genome sequences. As a result, important data about its genome organization and evolution were obtained, such as the lesser gene density of the large metacentric pair compared with the other metacentric chromosomes, which supports the theory of a sex proto-chromosome pair. In addition, chromosomes with a high number of linked genes that are conserved, even in distant species, were detected. This kind of result widens the knowledge of this species' chromosome dynamics and evolution.

Integrative genetic map of repetitive DNA in the sole Solea senegalensis genome shows a Rex transposon located in a proto-sex chromosome.

Repetitive sequences play an essential role in the structural and functional evolution of the genome, particularly in the sexual chromosomes. The Senegalese sole (Solea senegalensis) is a valuable flatfish in aquaculture albeit few studies have addressed the mapping and characterization of repetitive DNA families. Here we analyzed the Simple Sequence Repeats (SSRs) and Transposable elements (TEs) content from fifty-seven BAC clones (spanning 7.9 Mb) of this species, located in chromosomes by multiple fluorescence in situ hybridization (m-BAC-FISH) technique. The SSR analysis revealed an average density of 675.1 loci per Mb and a high abundance (59.69%) of dinucleotide coverage was observed, being 'AC' the most abundant. An SSR-FISH analysis using eleven probes was also carried out and seven of the 11 probes yielded positive signals. 'AC' probes were present as large clusters in almost all chromosomes, supporting the bioinformatic analysis. Regarding TEs, DNA transposons (Class II) were the most abundant. In Class I, LINE elements were the most abundant and the hAT family was the most represented in Class II. Rex/Babar subfamily, observed in two BAC clones mapping to chromosome pair 1, showed the longest match. This chromosome pair has been recently reported as a putative sexual proto-chromosome in this species, highlighting the possible role of the Rex element in the evolution of this chromosome. In the Rex1 phylogenetic tree, the Senegalese sole Rex1 retrotransposon could be associated with one of the four major ancient lineages in fish genomes, in which it is included O. latipes.

Evolution of the Proto Sex-Chromosome in Solea senegalensis.

Solea senegalensis is a flatfish belonging to the Soleidae family within the Pleuronectiformes order. It has a karyotype of 2n = 42 (FN = 60; 6M + 4 SM + 8 St + 24 T) and a XX/XY system. The first pair of metacentric chromosomes has been proposed as a proto sex-chromosome originated by a Robertsonian fusion between acrocentric chromosomes. In order to elucidate a possible evolutionary origin of this chromosome 1, studies of genomic synteny were carried out with eight fish species. A total of 88 genes annotated within of 14 BACs located in the chromosome 1 of S. senegalensis were used to elaborate syntenic maps. Six BACs (BAC5K5, BAC52C17, BAC53B20, BAC84K7, BAC56H24, and BAC48P7) were distributed in, at least, 5 chromosomes in the species studied, and a group of four genes from BAC53B20 (grsf1, rufy3, slc4a4 and npffr2) and genes from BAC48K7 (dmrt2, dmrt3, dmrt1, c9orf117, kank1 and fbp1) formed a conserved cluster in all species. The analysis of repetitive sequences showed that the number of retroelements and simple repeat per BAC showed its highest value in the subcentromeric region where 53B20, 16E16 and 48K7 BACs were localized. This region contains all the dmrt genes, which are associated with sex determination in some species. In addition, the presence of a satellite "chromosome Y" (motif length: 860 bp) was detected in this region. These findings allowed to trace an evolutionary trend for the large metacentric chromosome of S. senegalensis, throughout different rearrangements, which could be at an initial phase of differentiation as sex chromosome.

Genome and Phylogenetic Analysis of Genes Involved in the Immune System of Solea senegalensis - Potential Applications in Aquaculture.

Global aquaculture production continues to increase rapidly. One of the most important species of marine fish currently cultivated in Southern Europe is Solea senegalensis, reaching more than 300 Tn in 2017. In the present work, 14 Bacterial Artificial Chromosome (BAC) clones containing candidate genes involved in the immune system (b2m, il10, tlr3, tap1, tnfα, tlr8, trim25, lysg, irf5, hmgb2, calr, trim16, and mx), were examined and compared with other species using multicolor Fluorescence in situ Hybridization (mFISH), massive sequencing and bioinformatic analysis to determine the genomic surroundings and syntenic chromosomal conservation of the genomic region contained in each BAC clone. The mFISH showed that the groups of genes hmgb2-trim25-irf5-b2m; tlr3-lysg; tnfα-tap1, and il10-mx-trim16 were co-localized on the same chromosomes. Synteny results suggested that the studied BACs are placed in a smaller number of chromosomes in S. senegalensis that in other species. Phylogenetic analyses suggested that the evolutionary rate of immune system genes studied is similar among the taxa studied, given that the clustering obtained was in accordance with the accepted phylogenetic relationships among these species. This study contributes to a better understanding of the structure and function of the immune system of the Senegalese sole, which is essential for the development of new technologies and products to improve fish health and productivity.

A preliminary integrated genetic map distinguishes every chromosome pair and locates essential genes related to abiotic adaptation of Crassostrea angulata/gigas.

BACKGROUND: The re-sequencing of C. angulata has revealed many polymorphisms in candidate genes related to adaptation to abiotic stress that are not present in C. gigas; these genes, therefore, are probably related to the ability of this oyster to retain high concentrations of toxic heavy metals. There is, in addition, an unresolved controversy as to whether or not C. angulata and C. gigas are the same species or subspecies. Both oysters have 20 metacentric chromosomes of similar size that are morphologically indistinguishable. From a genomic perspective, as a result of the great variation and selection for heterozygotes in C. gigas, the assembly of its draft genome was difficult: it is fragmented in more than seven thousand scaffolds. RESULTS: In this work sixty BAC sequences of C. gigas downloaded from NCBI were assembled in BAC-contigs and assigned to BACs that were used as probes for mFISH in C. angulata and C. gigas. In addition, probes of H3, H4 histone, 18S and 5S rDNA genes were also used. Hence we obtained markers identifying 8 out the 10 chromosomes constituting the karyotype. Chromosomes 1 and 9 can be distinguished morphologically. The bioinformatic analysis carried out with the BAC-contigs annotated 88 genes. As a result, genes associated with abiotic adaptation, such as metallothioneins, have been positioned in the genome. The gene ontology analysis has also shown many molecular functions related to metal ion binding, a phenomenon associated with detoxification processes that are characteristic in oysters. Hence the provisional integrated map obtained in this study is a useful complementary tool for the study of oyster genomes. CONCLUSIONS: In this study 8 out of 10 chromosome pairs of Crassostrea angulata/gigas were identified using BAC clones as probes. As a result all chromosomes can now be distinguished. Moreover, FISH showed that H3 and H4 co-localized in two pairs of chromosomes different that those previously escribed. 88 genes were annotated in the BAC-contigs most of them related with Molecular Functions of protein binding, related to the resistance of the species to abiotic stress. An integrated genetic map anchored to the genome has been obtained in which the BAC-contigs structure were not concordant with the gene structure of the C. gigas scaffolds displayed in the Genomicus database.

Evidence for a Robertsonian fusion in Solea senegalensis (Kaup, 1858) revealed by zoo-FISH and comparative genome analysis.

BACKGROUND: Solea senegalensis (Kaup, 1858) is a commercially important flatfish species, belonging to the Pleuronectiformes order. The taxonomy of this group has long been controversial, and the karyotype of the order presents a high degree of variability in diploid number, derived from chromosomal rearrangements such as Robertsonian fusions. Previously it has been proposed that the large metacentric chromosome of S. senegalensis arises from this kind of chromosome rearrangement and that this is a proto-sex chromosome. RESULTS: In this work, the Robertsonian origin of the large metacentric chromosome of S. senegalensis has been tested by the Zoo-FISH technique applied to two species of the Soleidae family (Dicologlossa cuneata and Dagetichthys lusitanica), and by comparative genome analysis with Cynoglossus semilaevis. From the karyotypic analysis we were able to determine a chromosome complement comprising 2n = 50 (FN = 54) in D. cuneata and 2n = 42 (FN = 50) in D. lusitanica. The large metacentric painting probe gave consistent signals in four acrocentric chromosomes of the two Soleidae species; and the genome analysis proved a common origin with four chromosome pairs of C. semilaevis. As a result of the genomic analysis, up to 61 genes were annotated within the thirteen Bacterial Artificial Chromosome clones analysed. CONCLUSIONS: These results confirm that the large metacentric chromosome of S. senegalensis originated from a Robertsonian fusion and provide new data about the chromosome evolution of S. senegalensis in particular, and of Pleuronectiformes in general.

Analysis of the histone cluster in Senegalese sole (Solea senegalensis): evidence for a divergent evolution of two canonical histone clusters.

The Senegalese sole (Solea senegalensis) is commercially very important and a priority species for aquaculture product diversification. The main histone cluster was identified within two BAC clones. However, two replacement histones (H1.0 and H3.3) were found in another BAC clone. Different types of canonical histones H2A and H2B were found within the same species for the first time. Phylogenetic analysis demonstrated that the different types of H1, H2A, and H2B histones were all more similar to each other than to canonical histones from other species. The canonical histone H3 of S. senegalensis differs from subtypes H3.1 and H3.2 in humans at the site of residue 96, where a serine is found instead of an alanine. This same polymorphism has been found only in Danio rerio. The karyotype of S. senegalensis comprises 21 pairs of chromosomes, distributed in 3 metacentric pairs, 2 submetacentric pairs, 4 subtelocentric pairs, and 12 acrocentric pairs. The two BAC clones that contain the clusters of canonical histones were both mapped on the largest metacentric pair, and mFISH analysis confirmed the co-location with the dmrt1 gene in that pair. Three chromosome markers have been identified which, in addition to those previously described, account for 18 chromosome pairs in S. senegalensis.

Evolutionary Dynamics of rDNAs and U2 Small Nuclear DNAs in Triportheus (Characiformes, Triportheidae): High Variability and Particular Syntenic Organization.

Multigene families correspond to a group of genes tandemly repeated, showing enormous diversity in both number of units and genomic organization. In fishes, unlike rDNAs that have been well explored in cytogenetic studies, U2 small nuclear RNA (snRNA) genes are poorly investigated concerning their chromosomal localization. All Triportheus species (Characiformes, Triportheidae) studied so far carry a ZZ/ZW sex chromosomes system, where the W chromosome contains a huge 18S rDNA cistron. In some species the syntenic organization of rDNAs on autosomes was also verified. To explore this particular organization, we performed three-color-fluorescence in situ hybridization using 5S, 18S rDNA, and U2 snRNA genes as probes in eight Triportheus species. This work represents the first one analyzing the chromosomal distribution of U2 snRNA genes in genomes of Triportheidae. The variability in number of rDNA clusters, and the divergent syntenies for these three multigene families, put in evidence their evolutionary dynamism, revealing a much more complex organization of these genes than previously supposed for closely related species. Our study also provides additional data on the accumulation of repetitive sequences in the sex-specific chromosome. Besides, the chromosomal organization of U2 snDNAs among fish species is also reviewed.

Integrated gene mapping and synteny studies give insights into the evolution of a sex proto-chromosome in Solea senegalensis.

The evolution of genes related to sex and reproduction in fish shows high plasticity and, to date, the sex determination system has only been identified in a few species. Solea senegalensis has 42 chromosomes and an XX/XY chromosome system for sex determination, while related species show the ZZ/ZW system. Next-generation sequencing (NGS), multi-color fluorescence in situ hybridization (mFISH) techniques, and bioinformatics analysis have been carried out, with the objective of revealing new information about sex determination and reproduction in S. senegalensis. To that end, several bacterial artificial chromosome (BAC) clones that contain candidate genes involved in such processes (dmrt1, dmrt2, dmrt3, dmrt4, sox3, sox6, sox8, sox9, lh, cyp19a1a, amh, vasa, aqp3, and nanos3) were analyzed and compared with the same region in other related species. Synteny studies showed that the co-localization of dmrt1-dmrt2-drmt3 in the largest metacentric chromosome of S. senegalensis is coincident with that found in the Z chromosome of Cynoglossus semilaevis, which would potentially make this a sex proto-chromosome. Phylogenetic studies show the close proximity of S. senegalensis to Oryzias latipes, a species with an XX/XY system and a sex master gene. Comparative mapping provides evidence of the preferential association of these candidate genes in particular chromosome pairs. By using the NGS and mFISH techniques, it has been possible to obtain an integrated genetic map, which shows that 15 out of 21 chromosome pairs of S. senegalensis have at least one BAC clone. This result is important for distinguishing those chromosome pairs of S. senegalensis that are similar in shape and size. The mFISH analysis shows the following co-localizations in the same chromosomes: dmrt1-dmrt2-dmrt3, dmrt4-sox9-thrb, aqp3-sox8, cyp19a1a-fshb, igsf9b-sox3, and lysg-sox6.

Adaptation to abiotic stress in the oyster Crassostrea angulata relays on genetic polymorphisms.

Here we describe the whole genome re-sequencing of the Portuguese oyster Crassostrea angulata, an edible cupped oyster of major commercial importance with an important role as biosensor of coastal water pollution. We sequenced the genome of the C. angulata to 29.3-fold coverage using ABI SOLID system. Comparisons of the sequences with the reference assembly of the Pacific oyster (Crassostrea gigas), yielded 129 million SNPs, 151,620 from which were located in 20,908 genes from the C. gigas database. The analysis of Gene Ontology (GO) terms associated with gene regions containing SNPs, revealed that significant GO terms showing differences between the two oyster species, were related to activities of response to stress caused both by drying and by metal contamination. In the Biological Process domain, the GO terms ion transport, phosphorylation and proteolysis processes, among others, showed many polymorphic genes in C. angulata. These processes are related to combating genotoxic and hypo-osmotic stress in the oyster. It is noteworthy that more than 200 polymorphic genes were associated with DNA repair processes. These results reveal that most of the gene polymorphisms observed in C. angulata are associated with processes related to genome adaptation to abiotic stress in estuarine regions and support that genetic polymorphisms may be the base to the observed ability of C. angulata to retain the phenomenally high concentrations of toxic heavy metals. Our results also provide the framework for future investigations to establish the molecular basis of phenotypic variation of adaptive traits and should contribute to the management of the species' genetic resources.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray.

BACKGROUND: Senegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution. RESULTS: A comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB. CONCLUSIONS: Transcriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

A rapid method for differentiating four species of the Engraulidae (anchovy) family.

The four species of the Engraulidae family: European anchovy (Engraulis encrasicolus), Californian anchovy (Engraulis mordax), Peruvian anchoveta (Engraulis ringens), and Japanese anchovy (Engraulis japonicus) studied in this work are very similar morphologically, and it is very difficult to distinguish between them, especially when frozen or processed. We have used the 5S rDNA as a molecular marker to discriminate these four species and used specific primers designed for each species in the nontranscribed spacers (NTS) of these genes. Multiplex PCR was performed with three pairs of primers, and three different sizes were obtained: 597 bp E. encrasicolus, 598 bp E. japonicus, 380 bp E. mordax, and 250 bp E. ringens. For the species E. encrasicolus and E. japonicus, PCR-RFLP was used as an additional technique to distinguish between them because their NTS sequences showed considerable similarity.

Expression profiling of the sex-related gene Dmrt1 in adults of the Lusitanian toadfish Halobatrachus didactylus (Bloch and Schneider, 1801).

Doublesex and mab-3 related transcription factor 1 (Dmrt1) gene is a widely conserved gene involved in sex determination and differentiation across phyla. To gain insights on Dmrt1 implication for fish gonad cell differentiation and gametogenesis development, its mRNA was isolated from testis and ovary from the Lusitanian toadfish (Halobatrachus didactylus). The cDNA from Dmrt1 was synthesized and cloned, whereas its quantitative and qualitative gene expression, as well as its protein immunolocalization, were analyzed. A main product of 1.38 kb, which encodes a protein of 295 aa, was reported, but other minority Dmrt1 products were also identified by RACE-PCR. This gene is predominantly expressed in testis (about 20 times more than in other organs or tissues), specially in spermatogonia, spermatocytes and spermatids, as well as in somatic Sertoli cells, indicating that Dmrt1 plays an important role in spermatogenesis. Although Dmrt1 transcripts also seem to be involved in oogenesis development, and it cannot be excluded that toadfish Dmrt1 could be functionally involved in other processes not related to sex.