Satellite DNAs (satDNAs) are tandemly repeated sequences that are usually located on the heterochromatin, and the entire collection of satDNAs within a genome is called satellitome. Primarily, these sequences are not under selective pressure and evolve by concerted evolution, resulting in elevated rates of divergence between the satDNA profiles of reproductive isolated species/populations. Here, we characterized two additional satellitomes of Characiformes fish (Colossoma macropomum and Piaractus mesopotamicus) that diverged approximately 30 million years ago, while still retaining conserved karyotype features. The results we obtained indicated that several satDNAs (50% of satellite sequences in P. mesopotamicus and 43% in C. macropomum) show levels of conservation between the analyzed species, in the nucleotide and chromosomal levels. We propose that long-life cycles and few genomic changes could slow down rates of satDNA differentiation.

Chromosomal rearrangements play a significant role in the evolution of fish genomes, being important forces in the rise of multiple sex chromosomes and in speciation events. Repetitive DNAs constitute a major component of the genome and are frequently found in heterochromatic regions, where satellite DNA sequences (satDNAs) usually represent their main components. In this work, we investigated the association of satDNAs with chromosome-shuffling events, as well as their potential relevance in both sex and karyotype evolution, using the well-known Pyrrhulina fish model. Pyrrhulina species have a conserved karyotype dominated by acrocentric chromosomes present in all examined species up to date. However, two species, namely P. marilynae and P. semifasciata, stand out for exhibiting unique traits that distinguish them from others in this group. The first shows a reduced diploid number (with 2n = 32), while the latter has a well-differentiated multiple X₁X₂Y sex chromosome system. In addition to isolating and characterizing the full collection of satDNAs (satellitomes) of both species, we also in situ mapped these sequences in the chromosomes of both species. Moreover, the satDNAs that displayed signals on the sex chromosomes of P. semifasciata were also mapped in some phylogenetically related species to estimate their potential accumulation on proto-sex chromosomes. Thus, a large collection of satDNAs for both species, with several classes being shared between them, was characterized for the first time. In addition, the possible involvement of these satellites in the karyotype evolution of P. marilynae and P. semifasciata, especially sex-chromosome formation and karyotype reduction in P. marilynae, could be shown.

Contrasting with most bird species that present an ancestral-like karyotype (with 2n = 80), the only extant Cariamidae birds, the Red-legged (Cariama cristata) and Black-legged (Chunga burmeisteri) Seriemas, have high 2n and atypically large Z chromosomes. This study combined cytogenetic, bioinformatic, and genomic analyses to examine the distinctive characteristics of an unusual bird karyotype, with a focus on repetitive elements and sex chromosomes. Whole-genome alignments and chromosomal painting with a Z-chromosome-specific probe were also performed against the emu (a species with an ancestral-like karyotype). The satellitomes of C. cristata and C. burmeisteri were composed of only four and 6 long satDNAs, respectively. These satDNAs showed similarity with other repetitive sequences, mostly transposable elements, and were mapped in the pericentromeric regions of several chromosome pairs. CcrSat02-1104 mostly covered the Z and W sex chromosomes, besides being spread throughout additional chromosomes. Interstitial telomeric sites were not detected, even in the Z chromosome, and none of the 16 microsatellites tested showed positive signals on the C. cristata chromosomes. The genome alignments showed that the karyotype evolution that occurred in C. cristata may have involved significant chromosomal reshuffling, particularly fissions. Notwithstanding certain internal inversions, the Z chromosome retained homology with that of the emu. However, repetitive sequences also accumulated on the Z chromosome, contributing to its enlargement relative to the pattern observed in ancestral avian groups.

The beetles of the subtribe Oedionychina (Chrysomelidae, Alticinae) are the only ones that have the atypical giant and achiasmatic sex chromosomes, which are substantially larger than the autosomes. Previous cytogenetic analyses suggest a large accumulation of repetitive DNA in the sex chromosomes. In this study, we examined the similarity of X and Y chromosomes in four Omophoita species and compared genomic differentiation to better understand the evolutionary process and the giant sex chromosomes origin. Intraspecific genomic comparation using male and female genomes of O. octoguttata and interespecific analyses using genomic DNA of O. octoguttata, O. sexnotata, O. magniguttis, and O. personata were performed. In addition, whole chromosome painting (WCP) experiments were performed with X and Y chromosome probes of O. octogutatta. CGH analysis revealed great genomic similarity between the sexes and a sex-specific region on the Y chromosome, and interspecific analysis revealed a genomic divergence between species. In contrast, WCP results revealed that the sex chromosomes of O. octoguttata have high intra- and interspecific similarity with the studied species. Our data support a common origin under the canonical evolution of the sex chromosomes in this group, as they have high genomic similarity between them.

A few species have evolved multiple sex chromosome systems with more than two Xs or Ys due to sex chromosome-autosome translocations. Among vertebrates, frogs (Anura) have the highest known number of such neo-sex chromosome systems, making them interesting for studying how such systems evolve. In this work, we investigated two Leptodactylus species, L. pentadactylus (LPE) and L. paraensis (LPA), with large ring multivalents in male meiosis, using genomic and cytogenetic investigation of repetitive DNA sequences, including satellite DNAs (satDNAs), and transposable elements (TEs). SatDNA mapping identify individual chromosomes in the LPE ring, and morphologies suggest that all chromosomes are shared with the LPA ring although a common ring origin is not firmly supported. In situ mapping suggests recent satDNA accumulation in subtelomeric regions since the split from the outgroups, likely unrelated to the translocations that created sex-linkage, which probably involved breaks in the pericentromeric regions.

This methodological study outlines effective and standardized procedures for obtaining high-quality chromosomal preparations in birds, which display complex karyotypes with a large number of microchromosomes. We describe every stage from sample obtaining and culture setup to the successful acquisition of metaphase chromosomes appropriate for in-depth analysis by combining three main methodologies (fibroblast, lymphocyte, and bone marrow cultures) into a coherent and methodical workflow. Furthermore, we offer thorough troubleshooting instructions to tackle typical problems, such as low mitotic indices, poor chromosome spreading, contamination, and suboptimal chromosomal morphology. We also focused on repeatability and adaptation across several bird taxa, taking into consideration species-specific differences in their body sizes, cell development, and chromosomal behavior, extending beyond standard procedure improvement. The incorporation of diverse methodologies into a unified framework offers avian cytogenetics researchers a readily available and practical workflow. Ultimately, our approach enhances the integration of cytogenetic data with novel genomic resources, facilitates the re-evaluation of chromosomal counts, and enables more precise karyotype characterization. This integration is crucial for advancing our understanding of chromosomal evolution, avian genome architecture, and the broader mechanisms underlying avian speciation and diversification.