BackgroundThe fish, Erythrinus erythrinus, shows an interpopulation diversity, with four karyomorphs differing by chromosomal number, chromosomal morphology and heteromorphic sex chromosomes. Karyomorph A has a diploid number of 2n = 54 and does not have differentiated sex chromosomes. Karyomorph D has 2n = 52 chromosomes in females and 2n = 51 in males, and it is most likely derived from karyomorph A by the differentiation of a multiple X1X2Y sex chromosome system. In this study, we analyzed karyomorphs A and D by means of cytogenetic approaches to evaluate their evolutionary relationship.ResultsConspicuous differences in the distribution of the 5S rDNA and Rex3 non-LTR retrotransposon were found between the two karyomorphs, while no changes in the heterochromatin and 18S rDNA patterns were found between them. Rex3 was interstitially dispersed in most chromosomes. It had a compartmentalized distribution in the centromeric regions of only two acrocentric chromosomes in karyomorph A. In comparison, in karyomorph D, Rex3 was found in 22 acrocentric chromosomes in females and 21 in males. All 5S rDNA sites co-localized with Rex3, suggesting that these are associated in the genome. In addition, the origin of the large metacentric Y chromosome in karyomorph D by centric fusion was highlighted by the presence of internal telomeric sites and 5S rDNA/Rex3 sites on this chromosome.ConclusionWe demonstrated that some repetitive DNAs (5S rDNA, Rex3 retroelement and (TTAGGG)n telomeric repeats) were crucial for the evolutionary divergence inside E. erythrinus. These elements were strongly associated with the karyomorphic evolution of this species. Our results indicate that chromosomal rearrangements and genomic modifications were significant events during the course of evolution of this fish. We detected centric fusions that were associated with the differentiation of the multiple sex chromosomes in karyomorph D, as well as a surprising increase of associated 5S rDNA/Rex3 loci, in contrast to karyomorph A. In this sense, E. erythrinus emerges as an excellent model system for better understanding the evolutionary mechanisms underlying the huge genome diversity in fish. This organism can also contribute to understanding vertebrate genome evolution as a whole.

Fish exhibit the greatest diversity of all vertebrates, making this group extremely attractive for the study of a number of evolutionary questions. Fish genomes have intrinsic characteristics that may be responsible for the amazing diversity of fish species observed, but little is known about their structure and organization. A large amount of data from mapping of repetitive DNA sequences of several species has been generated, providing an important source of information for better understanding the involvement of repetitive DNA sequences in chromosomal organization. Almost all classes of repeated DNAs have been mapped in fishes, and all fish genomes analyzed contain at least one, mostly all types of repetitive DNAs. DNA sequence data combined with the chromosomal mapping of these repeated elements by means of cytogenetic techniques can provide a clearer picture of the genome, which is not yet clearly defined, even if already sequenced. In this chapter, we do not aim to analyze all available data on the chromosomal distribution of repetitive DNAs in fish species, but instead wish to draw attention to the impact of repetitive DNA sequences on fish karyotyping and genome evolution, with a particular focus on B chromosome origin and maintenance and on the differentiation of sex chromosomes. We also discuss the integration of chromosome analysis and genomic data, which represents a promising tool for fish cytogenomics.

The neotropical fish, Hoplias malabaricus, is well known for its population-specific karyotypic diversity and the variation of its sex chromosomes. Seven karyomorphs (A to G) have been previously described with an XY, X 1 X 2 Y and XY 1 Y 2 sex chromosome system found in karyomorphs B, D and G, respectively. We compared the chromosomal characteristics of karyomorphs C and D using C-banding, staining with CMA 3 and DAPI, and by mapping the location of 18S rDNA, 5SHindIII-DNA and (TTAGGG) n repeat sequences. Our results show conserved karyotypes in both karyomorphs, a nascent XX/XY sex chromosome system in karyomorph C and the origin of neo-Y chromosome in karyomorph D. The X and Y chromosomes of karyomorph C differ only slightly because of the amplification of repetitive sequences on the X chromosome, resulting in a homomorphic condition in all females and a heteromorphic condition in all males examined. Our study showed that chromosomes X and 20 of karyomorph C have similar patterns to the X 1 and X 2 chromosomes of karyomorph D, and are probably homologous. We showed that the neo-Y chromosome of karyomorph D shares similar patterns to the chromosomes Y and 20 of karyomorph C, and probably evolved through tandem fusion between Ypter/20pter. An interstitial site of the satellite 5SHindIII-DNA on the neo-Y reinforces the hypothesized dicentric nature of this chromosome. Our study shows the initial steps in XY chromosome differentiation in H. malabaricus and, in a broader context, contributes to the understanding of the evolutionary pathway leading to a multiple X 1 X 2 Y sex chromosome system in fishes.

Karyotype and chromosomal characteristics from 3 allopatric populations of Hoplias malabaricus, cytogenetically the most studied Erythrinidae taxon, were investigated using different staining techniques (C-, Ag-, and CMA₃ banding) as well as fluorescent in situ hybridization (FISH) to detect 18S rDNA, 5S rDNA, and 5SHindIII satellite DNA sites. The isolation, cloning and characterization of an 18S rDNA probe from H. malabaricus genome were also performed for the first time in order to develop a more specific probe. The 3 populations, named PR, CR, and DR, showed identical karyotypes, with 2n = 42 chromosomes composed of 11 m pairs and 10 sm pairs, without heteromorphic sex chromosomes, which characterize the populations as belonging to karyomorph A. In all populations C-positive heterochromatin was situated in the centromeric/pericentromeric regions of the chromosomes, as well as in the telomeric region of several pairs. A conspicuous proximal heterochromatic block on the long arm of pair No. 16 was the only GC-rich segment in the karyotypes. 5SHindIII satellite DNA was always mapped in the centromeric region of several chromosomes. The 18S rDNA sites were situated on the telomeric or centromeric regions, whereas the 5S rDNA showed an interstitial or proximal location in some pairs. Several chromosomes bearing these repetitive DNA sequences were shared by the 3 populations, alongside with some exclusive chromosomal markers. In this sense, population CR was the most differentiated one, including a syntenic condition for the 18S and 5S rDNA probes, as confirmed by double FISH. Thus, despite their inclusion in the same major karyotypic group, the distinct populations cannot be considered an absolute evolutionary unit, as evidenced by their inner chromosomal differentiations.

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C(0)t-1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII-DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY – karyomorph B and a multiple X₁X₁X₂X₂/X₁X₂Y – karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.

Background: Seven karyomorphs of the fish, Hoplias malabaricus (A-G) were previously included in two major groups, Group I (A, B, C, D) and Group II (E, F, G), based on their similar karyotype structure. In this paper, karyomorphs from Group I were analyzed by means of distinct chromosomal markers, including silver-stained nucleolar organizer regions (Ag-NORs) and chromosomal location of repetitive sequences (18S and 5S rDNA, and satellite 5SHindIII-DNA), through fluorescence in situ hybridization (FISH), in order to evaluate the evolutionary relationships among them.

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