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.

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.

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.

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.

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.

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.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles.

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

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.

Assessment of tools for marker-assisted selection in a marine commercial species: significant association between MSTN-1 gene polymorphism and growth traits.

Growth is a priority trait from the point of view of genetic improvement. Molecular markers linked to quantitative trait loci (QTL) have been regarded as useful for marker-assisted selection in complex traits as growth. Polymorphisms have been studied in five candidate genes influencing growth in gilthead seabream (Sparus aurata): the growth hormone (GH), insulin-like growth factor-1 (IGF-1), myostatin (MSTN-1), prolactin (PRL), and somatolactin (SL) genes. Specimens evaluated were from a commercial broodstock comprising 131 breeders (from which 36 males and 44 females contributed to the progeny). In all samples eleven gene fragments, covering more than 13,000 bp, generated by PCR-RFLP, were analyzed; tests were made for significant associations between these markers and growth traits. ANOVA results showed a significant association between MSTN-1 gene polymorphism and growth traits. Pairwise tests revealed several RFLPs in the MSTN-1 gene with significant heterogeneity of genotypes among size groups. PRL and MSTN-1 genes presented linkage disequilibrium. The MSTN-1 gene was mapped in the centromeric region of a medium-size acrocentric chromosome pair.

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.

Molecular characterization and evolution of an interspersed repetitive DNA family of oysters.

When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150 bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268 bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.

Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate.

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.