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.

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.

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.

Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227- and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.

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The 5S ribosomal DNA (rDNA) consists of one transcriptional unit of about 120 base pairs, which is separated from the next unit by a non-transcribed spacer (NTS). The coding sequence and the NTS together form a repeat unit which can be found in hundreds to thousands of copies tandemly repeated in the genomes. The NTS regions seem to be subject to rapid evolution. The first general model of evolution of these multigene families was referred to as divergent evolution, based on studies using hemoglobin and myoglobin as model systems. Later studies showed that nucleotide sequences of different multigene family members are more closely related within species than between species. This observation led to a new model of multigene family evolution, termed concerted evolution. Another model of evolution, named the birth-and-death model, has been found to be more suitable to explain the long-term evolution of these multigene families. According to this model, new genes originate by successive duplications, and these new genes are either maintained for a long time or are lost, or else degenerate into pseudogenes. In this review we describe different sources of variability in the 5S rDNA genes observed in several distinct fish species. This variability is mainly referred to NTSs and includes the presence of other multigene families (mainly LINEs, SINEs, non-LTR retrotransposons, and U snRNA families). Different types of microsatellites have also been found to contribute to the increase of variability in this region. Our recent results suggest that horizontal transfer contributes to the increase of diversity in the NTSs of some species. Variability in the 5S rDNA coding region affecting the stability of the structure, but without effects on the function of the 5S rRNA, is also described. Retrotransposons seem to be responsible for the high dynamism of 5S rDNA, while microsatellites acting as recombination hot spots could stabilize a wide variety of unusual DNA structures, affecting DNA replication and enhancing or decreasing promoter activity in gene expression. The relationship between the high variability found at molecular level and the low variability found at chromosomal level is also discussed.

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA)n, and (TTAGGG)n, and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG)n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA)n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.

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.