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.