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.

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.

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.

The 5S rDNA high dynamism in Diplodus sargus is a transposon-mediated mechanism. Comparison with other multigene families and Sparidae species.

There has been considerable discussion in recent years on the evolution of the tandemly repeated multigene families, since some organisms show a concerted model whereas others show a birth-and-death model. This controversial subject extends to several species of fish. In this study, three species of the Sparidae family (Pagrus pagrus, P. auriga and Diplodus sargus) and an interspecific hybrid (P. pagrus (♀) × P. auriga (♂)) have been studied at both molecular and cytogenetic level, taking three different multigene families (5S rDNA, 45S rDNA and U2 snDNA). Results obtained with the 5S rDNA in P. pagrus and P. auriga are characterized by a considerable degree of conservation at the two levels; however, an extraordinary variation was observed in D. sargus at the two levels, which has never been found in other fishes studied to date. As a consequence of this, the evolutionary model of the multigene families is discussed considering the results obtained and others from the bibliography. The result obtained in the hybrid allowed the recombination frequency in each multigene family to be estimated.

Evidence for 5S rDNA horizontal transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families.

BACKGROUND: The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). RESULTS: Two types of 5S rDNA were observed, named type α and type ß. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type ß specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. CONCLUSIONS: A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.

Genetic characterization of Plectorhinchus mediterraneus yields important clues about genome organization and evolution of multigene families.

BACKGROUND: Molecular and cytogenetic markers are of great use for to fish characterization, identification, phylogenetics and evolution. Multigene families have proven to be good markers for a better understanding of the variability, organization and evolution of fish species. Three different tandemly-repeated gene families (45S rDNA, 5S rDNA and U2 snDNA) have been studied in Plectorhinchus mediterraneus (Teleostei: Haemulidae), at both molecular and cytogenetic level, to elucidate the taxonomy and evolution of these multigene families, as well as for comparative purposes with other species of the family. RESULTS: Four different types of 5S rDNA were obtained; two of them showed a high homology with that of Raja asterias, and the putative implication of a horizontal transfer event and its consequences for the organization and evolution of the 5S rDNA have been discussed. The other two types do not resemble any other species, but in one of them a putative tRNA-derived SINE was observed for the first time, which could have implications in the evolution of the 5S rDNA. The ITS-1 sequence was more related to a species of another different genus than to that of the same genus, therefore a revision of the Hamulidae family systematic has been proposed. In the analysis of the U2 snDNA, we were able to corroborate that U2 snDNA and U5 snDNA were linked in the same tandem array, and this has interest for tracing evolutionary lines. The karyotype of the species was composed of 2n = 48 acrocentric chromosomes, and each of the three multigene families were located in different chromosome pairs, thus providing three different chromosomal markers. CONCLUSIONS: Novel data can be extracted from the results: a putative event of horizontal transfer, a possible tRNA-derived SINE linked to one of the four 5S rDNA types characterized, and a linkage between U2 and U5 snDNA. In addition, a revision of the taxonomy of the Haemulidae family has been suggested, and three cytogenetic markers have been obtained. Some of these results have not been described before in any other fish species. New clues about the genome organization and evolution of the multigene families are offered in this study.

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family.

In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)(n) and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)(n) probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.

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.

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.

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.

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.

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.