Cytogenetic markers as a tool for characterization of hybrids of Astyanax Baird & Girard, 1854 and Hyphessobrycon Eigenmann, 1907.

Astyanax Baird et Girard, 1854, is one of the largest genera in the family Characidae and comprises 177 valid species. This genus has been the focus of cytogenetic studies primarily owing to the presence of B chromosomes and high karyotypic diversity among different populations. The intense genetic variability in Astyanax is one of the factors responsible for the occurrence of species complexes, which are groups (1) with certain difficulties in establishing common genetic pools or (2) belonging to different cryptic species. To evaluate cytogenetic marker inheritance and the possibility of the identification of these hybrids, this study aimed to describe cytogenetic hybrids from three strains of species of the genera Astyanax and Hyphessobrycon Eigenmann, 1908. A. lacustris Lütken, 1875, A. schubarti Britski, 1964, A. fasciatus Cuvier, 1819, and H. anisitsi Eigenmann, 1907 were used to generate three hybrid lineages. The diploid number, heterochromatin sites, and ribosomal genes (18S and 5S rDNA) of the parental strains and the hybrids were analyzed. The results indicated that the three hybrid lineages had cytogenetic markers of both parents, presenting Mendelian inheritance. However, differences in distribution of heterochromatic blocks were observed between the hybrids and the parent strains. Our results allowed the identification of the hybrid strains based on the cytogenetic markers applied, reinforcing the efficiency of cytogenetic markers as tools for identification and indicating that such events may increase the karyotypic diversity in the genera Astyanax and Hyphessobrycon.

Publisher Correction: High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Corrigenda: Cytogenetic markers as a tool for characterization of hybrids of Astyanax Baird & Girard, 1854 and Hyphessobrycon Eigenmann, 1907. Comparative Cytogenetics 14(2): 231-242. https://doi.org/10.3897/CompCytogen.v14i2.49513.

[This corrects the article DOI: 10.3897/CompCytogen.v14i2.49513.].

High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax.

The high-throughput analysis of satellite DNA (satDNA) content, by means of Illumina sequencing, unveiled 45 satDNA families in the genome of Astyanax paranae, with repeat unit length (RUL) ranging from 6 to 365 bp and marked predominance of short satellites (median length = 59 bp). The analysis of chromosomal location of 35 satDNAs in A. paranae, A. fasciatus and A. bockmanni revealed that most satellites are shared between the three species and show highly similar patterns of chromosome distribution. The high similarity in satellite DNA content between these species is most likely due to their recent common descent. Among the few differences found, the ApaSat44-21 satellite was present only on the B chromosome of A. paranae, but not on the A or B chromosomes of the two other species. Likewise, the ApaSat20-18 satellite was B-specific in A. paranae but was however present on A and B chromosomes of A. fasciatus and A. bockmanni. The isochromosome nature of B chromosomes in these species was evidenced by the symmetric location of many satDNAs on both B chromosome arms, and the lower symmetry observed in the A. fasciatus BfMa chromosome suggests that it is older than those analyzed in A. paranae and A. bockmanni.

Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting.

Eukaryote genomes are frequently burdened with the presence of supernumerary (B) chromosomes. Their origin is frequently investigated by chromosome painting, under the hypothesis that sharing the repetitive DNA sequences contained in the painting probes is a sign of common descent. However, the intragenomic mobility of many anonymous DNA sequences contained in these probes (e.g., transposable elements) adds high uncertainty to this conclusion. Here we test the validity of chromosome painting to investigate B chromosome origin by comparing its results for seven B chromosome types in two fish species genus Astyanax, with those obtained (1) by means of the physical mapping of 18S ribosomal DNA (rDNA), H1 histone genes, the As51 satellite DNA and the (AC)15 microsatellite, and (2) by comparing the nucleotide sequence of one of these families (ITS regions from ribosomal DNA) between genomic DNA from B-lacking individuals in both species and the microdissected DNA from two metacentric B chromosomes found in these same species. Intra- and inter-specific painting suggested that all B chromosomes that were assayed shared homologous DNA sequences among them, as well as with a variable number of A chromosomes in each species. This finding would be consistent with a common origin for all seven B chromosomes analyzed. By contrast, the physical mapping of repetitive DNA sequences failed to give support to this hypothesis, as no more than two B-types shared a given repetitive DNA. Finally, sequence analysis of the ITS regions suggested that at least some of the B chromosomes could have had a common origin.

First Description of supernumerary Chromosomes in Ictaluruspunctatus Rafinesque 1818 Reveals Active Ribosomal Genes in the B Complement.

The North American channel catfish Ictalurus punctatus Rafinesque 1818 is cultivated in the United States, Asia and Brazilian fish farms, and also utilized as a model species in aquaculture and genetic studies. In this work, cytogenetic analysis of . punctatus from Brazilian aquaculture revealed for the first time the presence of extra elements (supernumerary or B chromosomes) in this species. These elements were characterized as dot-like micro B chromosomes and were found in three individuals (varying from 0 to 1) and in one individual with higher incidence per cell (varying from 0 to 5; mean number of Bs per cell = 2.01). More specific cytogenetic techniques in this individual revealed 58 A chromosomes (standard complement) containing heterochromatic bands in the centromeric regions, a single Ag-NOR in a subtelocentric pair (also positive for 18S rDNA using the FISH technique) and multiple 5S rDNA clusters in three different subtelocentric chromosomes. Four B chromosomes were entirely Ag-NOR positive (also fully heterochromatic) and three presented 18S rDNA clusters by FISH. The occurrence of Ag-NOR and 18S ribosomal genes in both A and B chromosome complements may indicate an intraspecific origin for these extra chromosomes. Additionally, the terminal location of 18S ribosomal clusters in the Ag-NOR-bearing chromosomes and the presence of active NOR in the B chromosomes suggested that breakage events may be related to a possible recent origin of these extra elements. We suggest this data may be useful as cytogenetic information for future elucidation of the composition, origin and evolution of extra chromosomes in fishes.

New insights into karyotypic relationships among populations of Astyanax bockmanni (Teleostei, Characiformes) of different watersheds.

The fish constitute about 50% of all vertebrates, including a wide morphological and biological diversity, where the genus Astyanax is the most common and diverse, as described in virtually all freshwater environments. By occupying a basal position in the phylogeny of vertebrates, fish are an extremely favorable group for cytogenetic and evolutionary studies. The karyotype found in genus Astyanax diversity may involve a number of polymorphisms, which may be related to ploidy and karyotypic macrostructure, presence of B chromosomes, heterochromatin polymorphisms, and location of ribosomal genes. Nevertheless, the relationship between populations of this species is still poorly studied. Thus, the present work aimed to investigate karyotype variation, chromosomal relationships, and the behavior of 5S and 18S ribosomal genes in six populations of Astyanax bockmanni. The results confirmed the diploid number of 50 chromosomes in all the populations sampled, with the occurrence of one supernumerary chromosome in just one of them. In addition, all populations showed divergent patterns of constitutive heterochromatin and repetitive nucleolar sites. The fluorescence in situ hybridization (FISH) technique using 5S and 18S rDNA probes revealed distinct patterns of distribution for these conserved genes, while 5S rDNA genes were found located in two chromosome pairs, the 18S genes showed multiple marks dispersed in the genome characterizing an inter and intraindividual polymorphic behavior, as previously reported to occur with the utilization of the Ag-NOR technique. Thus, besides minor modifications observed in chromosome morphology, the populations of A. bockmanni analyzed revealed a preserved macrostructural feature, especially concerning to the diploid number; on the other hand, differences in microstructural characteristics indicated by the nucleolus organizer region (NOR) location, constitutive heterochromatin patterns, and distribution of ribosomal genes along the genome were clearly evident in the populations from different river basins, even located at short distances.

Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes).

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): dispersive location, association and co-localization in the genome.

Repetitive DNA sequences constitute a great portion of the genome of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Aiming to contribute to the knowledge of chromosome structure and organization of some repetitive DNA classes in the fish genome, chromosomes of two allopatric populations of Astyanax bockmanni were analyzed using classic cytogenetics techniques and fluorescent in situ hybridization, with probes for ribosomal DNA sequences, histone DNA and transposable elements. These Astyanax populations showed the same diploid number (2n = 50), however with differences in chromosome morphology, distribution of constitutive heterochromatin, and location of 18S rDNA and retroelement Rex3 sites. In contrast, sites for 5S rDNA and H1, H3 and H4 histones showed to be co-located and highly conserved. Our results indicate that dispersion and variability of 18S rDNA and heterochromatin sites are not associated with macro rearrangements in the chromosome structure of these populations. Similarly, distinct evolutionary mechanisms would act upon histone genes and 5S rDNA, contributing to chromosomal association and co-location of these sequences. Data obtained indicate that distinct mechanisms drive the spreading of repetitive DNAs in the genome of A. bockmanni. Also, mobile elements may account for the polymorphism of the major rDNA sites and heterochromatin in this genus.