Distribution Of Repetitive Sequences Research Articles

Multi-Chromosomal mitochondrial genome of medicinal plant Acorus tatarinowii (Acoraceae): Firstly reported from Acorales Order

Acorus tatarinowii Schott (A. tatarinowii), a well-known traditional Chinese medicinal plant renowned for its high medicinal value, but its mitochondrial genome (mitogenome) is still unexplored. In this study, we meticulously assembled the complete mitochondrial genome of A. tatarinowii using a combination of Illumina short reads and Oxford Nanopore long reads. Our findings revealed that A. tatarinowii possesses a complex chromosomal structural mitogenome, comprising two linear chromosomes and seven circular chromosomes. This mitogenome spans 1.81 Mb in length with a GC content of 38.29 %. Notably, it contained 24 unique mitochondrial core genes, seven unique variable genes, 17 tRNA genes, and three rRNA genes. Analyses of codon usage, most protein-coding genes (PCGs) exhibited a common codon usage preference, with RSCU values greater than 1, and the codon with the highest RSCU value was UAA(End, 1.90). We conducted a thorough analysis of repeat sequences, the distribution of repetitive sequences in nine mitochondrial chromosomes showed distinct patterns. Moreover, we identified 82 and 12 homologous fragments by comparing the sequences of chloroplast and nuclear genomes to the A. tatarinowii mitogenome, respectively. Lastly, We predicted a total of 234 potential RNA editing sites in 28 unique PCGs and discovered that the nad4 gene has been edited the most often, at 26 times. Our results contribute to the enrichment of mitochondrial genome resources for Acoraceae, and the mitogenome also can be used as a reference for other species.

Evolutionary tracks of chromosomal diversification in Trichopsis (Anabantiformes, Osphronemidae) fishes: New insights from a molecular cytogenetic perspective

Abstract. Mingkwan B, Sassi FDMC, Muanglenm N, Pinmongkhonkul S, Pinthong K, Tongnunui S, Yeesin P, Tanomtong A, Liehr T, Cioffi MDB, Supiwong W. 2023. Evolutionary tracks of chromosomal diversification in Trichopsis (Anabantiformes, Osphronemidae) fishes: New insights from a molecular cytogenetic perspective. Biodiversitas 24: 1551-1559. This study investigated the chromosomes of three Trichopsisspecies (namely Trichopsis pumila, Trichopsis vittata, and Trichopsis schalleri) using conventional (Giemsa stain, Ag-NOR) and molecularcytogenetic techniques. Fluorescence in situ hybridization (FISH) with repetitive DNAs, including 5S and 18S rDNAs, and microsatellites as probes were also performed. Our results indicated a conserved diploid number of 46 (2n) for all analyzed species of both sexes, although varying in their karyotype structure. Furthermore, T. schalleri and T. vittata karyotypes were presented with only acrocentric chromosomes (46a), while T. pumila had a submetacentric pair in addition to acrocentric ones (2sm+44a). Positive Ag-NOR sites were differentially found in each species adjacent to the acrocentric chromosome's centromeric and/or interstitial sub-centromeric regions. Both 5S and 18S rDNAs vary between the analyzed species, but all were localized in multiple sites. However, the distribution pattern of microsatellites differed in each species, mostly scattering at peri-centromeric and telomeric regions. These findings show that Trichopsis species, while having a conserved diploid number, differ significantly in the distribution of repetitive sequences in their karyotypes, particularly for rDNAs. These species-specific patterns can be useful for characterizing and identifying further different species and examining the evolutionary mechanisms that drove the evolution of these fishes' karyotypes.

Plant Cytogenetics: From Chromosomes to Cytogenomics.

Chromosomes have been studied since the late nineteenth century in the disciplines of cytology and cytogenetics. Analyzing their numbers, features, and dynamics has been tightly linked to the technical development of preparation methods, microscopes, and chemicals to stain them, with latest continuing developments described in this volume. At the end of the twentieth and beginning of the twenty-first centuries, DNA technology, genome sequencing, and bioinformatics have revolutionized how we see, use, and analyze chromosomes. The advent of in situ hybridization has shaped our understanding of genome organization and behavior by linking molecular sequence information with the physical location along chromosomes and genomes. Microscopy is the best technique to accurately determine chromosome number. Many features of chromosomes in interphase nuclei or pairing and disjunction at meiosis, involving physical movement of chromosomes, can only be studied by microscopy. In situ hybridization is the method of choice to characterize the abundance and chromosomal distribution of repetitive sequences that make up the majority of most plant genomes. These most variable components of a genome are found to be species- and occasionally chromosome-specific and give information about evolution and phylogeny. Multicolor fluorescence hybridization and large pools of BAC or synthetic probes can paint chromosomes and we can follow them through evolution involving hybridization, polyploidization, and rearrangements, important at a time when structural variations in the genome are being increasingly recognized. This volume discusses many of the most recent developments in the field of plant cytogenetics and gives carefully compiled protocols and useful resources.

Cytogenetic Analysis in the Toad Species Bufo spinosus, Bufotes viridis and Epidalea calamita (Anura, Bufonidae) from the Mediterranean Area.

Taxonomy in Bufonidae witnessed notable transformations. Bufotes viridis and Epidalea calamita, previously included in genus Bufo, were relocated in other genera, while the genus Bufo was restricted to members of the earlier Bufo bufo group. On the other hand, Bufo bufo sensu lato now includes four species: Bufo bufo, Bufo spinosus, Bufo verrucosissimus and Bufo eichwaldi. In this study, we examined three species of three Bufonidae genera (B. spinosus, B. viridis and E. calamita) by conventional (C-banding and Ag-NOR staining) and molecular (in situ hybridization with probes for telomeric repeats and rDNA loci, and genomic in situ hybridization (GISH)) cytogenetic methods. C-banding patterns are reported for the first time for B. spinosus and E. calamita populations from Iberian Peninsula and for B. viridis from Greece, and reveal several differences with the reported C-banded karyotypes described for other European populations of these species. Silver staining shows size heteromorphisms of the signals at the Nucleolar Organizing Region (NOR). By contrast, FISH with ribosomal probes only reveal size heteromorphism of rDNA sequences in E. calamita, suggesting that the differences observed after silver staining in B. spinosus and B. viridis should be attributed to differences in chromosomal condensation and/or gene activity rather than to differences in the copy number for ribosomal genes. Regarding telomeric repeats, E. calamita is the only species with interstitial telomeric sequences (ITS) located on centromeric regions, probably originated by accumulation of telomeric sequences in the centromeric heterochromatin. Finally, we analyzed the composition and distribution of repetitive sequences by genome in situ hybridization. These experiments reveal the accumulation of repetitive sequences in centromeric regions of the three species, although these sequences are not conserved when species from different genera are compared.

Repetitive Sequence Distribution on Saguinus, Leontocebus and Leontopithecus Tamarins (Platyrrhine, Primates) by Mapping Telomeric (TTAGGG) Motifs and rDNA Loci.

Tamarins are a distinct group of small sized New World monkeys with complex phylogenetic relationships and poorly studied cytogenetic traits. In this study, we applied molecular cytogenetic analyses by fluorescence in situ hybridization with probes specific for telomeric sequences and ribosomal DNA loci after DAPI/CMA3 staining on metaphases from five tamarin species, namely Leontocebus fuscicollis, Leontopithecus rosalia, Saguinus geoffroyi, Saguinus mystax and Saguinus oedipus, with the aim to investigate the distribution of repetitive sequences and their possible role in genome evolution. Our analyses revealed that all five examined species show similar karyotypes, 2n = 46, which differ mainly in the morphology of chromosome pairs 16-17 and 19-22, due to the diverse distribution of rDNA loci, the amplification of telomeric-like sequences, the presence of heterochromatic blocks and/or putative chromosomal rearrangements, such as inversions. The differences in cytogenetic traits between species of tamarins are discussed in a comparative phylogenetic framework, and in addition to data from previous studies, we underline synapomorphies and apomorphisms that appeared during the diversification of this group of New World monkeys.

Cytogenetic events in the endosperm of amphiploid Avena magna\u2009\xd7\u2009A. longiglumis

This study analysed cytogenetic events occurring in the syncytial endosperm of the Avena magna H. C. Murphy & Terrell × Avena longiglumis Durieu amphiploid, which is a product of two wild species having different genomes. Selection through the elimination of chromosomes and their fragments, including those translocated, decreased the level of ploidy in the endosperm below the expected 3n, leading to the modal number close to 2n. During intergenomic translocations, fragments of the heterochromatin-rich C-genome were transferred to the D and Al genomes. Terminal and non-reciprocal exchanges dominated, whereas other types of translocations, including microexchanges, were less common. Using two probes and by counterstaining with DAPI, the A. longiglumis and the rare exchanges between the D and Al genomes were detected by GISH. The large discontinuity in the probe labelling in the C chromosomes demonstrated inequality in the distribution of repetitive sequences along the chromosome and probable intragenomic rearrangements. In the nucleus, the spatial arrangement of genomes was non-random and showed a sectorial-concentric pattern, which can vary during the cell cycle, especially in the less stable tissue like the hybrid endosperm.

Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae).

Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

Alteration of active and repressive histone marks during adipogenic differentiation of porcine mesenchymal stem cells

A characteristic spatial distribution of the main chromatin fractions is observed in most mammalian cell nuclei, with euchromatin localized in the interior and heterochromatin at the nuclear periphery. It has been shown that interactions of heterochromatin with the nuclear lamina are necessary to establish this conventional architecture. Adipocytes are specific cells in which a reduction in lamin A/C expression is observed. We hypothesize that the loss of lamin A/C during adipogenic differentiation of mesenchymal stem cells (MSCs) may be associated with the reorganization of the main classes of chromatin in the nucleus. Thus, in this study, we examine the abundance and nuclear distribution of selected heterochromatin (H3K9me3, H3K27me3 and H4K20me3) and euchromatin (H4K8ac, H3K4me3 and H3K9ac) histone marks during in vitro adipogenesis, using the pig as a model organism. We found that not only did the expression of lamin A/C decrease in our differentiation system, but so did the expression of lamin B receptor (LBR). The level of two heterochromatin marks, H3K27me3 and H4K20me3, increased during differentiation, while no changes were observed for H3K9me3. The levels of two euchromatin histone marks, H4K8ac and H3K9ac, were significantly higher in adipocytes than in undifferentiated cells, while the level of H3K4me3 did not change significantly. The spatial distribution of all the examined histone marks altered during in vitro adipogenesis. H3K27me3 and H4K20me3 moved towards the nuclear periphery and H3K9me3 localized preferentially in the intermediate part of adipocyte nuclei. The euchromatin marks H3K9ac and H3K4me3 preferentially occupied the peripheral part of the adipocyte nuclei, while H4K8ac was more evenly distributed in the nuclei of undifferentiated and differentiated cells. Analysis of the nuclear distribution of repetitive sequences has shown their clustering and relocalization toward nuclear periphery during differentiation. Our study shows that dynamic changes in the abundance and nuclear distribution of active and repressive histone marks take place during adipocyte differentiation. Nuclear reorganization of heterochromatin histone marks may allow the maintenance of the nuclear morphology of the adipocytes, in which reduced expression of lamin A/C and LBR is observed.

Clusters of Repetitive DNA Sequences in Chromosomes of Voles of the Subgenus Microtus (Microtus, Arvicolidae)

The study is focused on the analysis of the mechanisms underlying the formation and distribution of repeat clusters in mammalian chromosomes, as exemplified by a group of closely related species of voles of the subgenus Microtus (Microtus, Arvicolini). The distribution of repetitive sequences that are the parts of de novo formed heterochromatic regions of Microtus arvalis in two chromosomal forms of this species and three closely related species, M. rossiaemeridionalis, M. kirgisorum, and M. transcaspicus, was analyzed in detail. Possible relationships between the introduction of repetitive sequences, their transpositions, amplification, and the formation of reproductive isolation, which can lead to speciation, are discussed.

Karyotype Evolution and Distinct Evolutionary History of the W Chromosomes in Swallows (Aves, Passeriformes)

As in many other bird groups, data on karyotype organization and distribution of repetitive sequences are also lacking in species belonging to the family Hirundinidae. Thus, in the present study, we analyzed the karyotypes of 3 swallow species (Progne tapera, Progne chalybea, and Pygochelidon cyanoleuca) by Giemsa and AgNOR staining, C-banding, and FISH with 11 microsatellite sequences. The diploid chromosome number was 2n = 76 in all 3 species, and NORs were observed in 2 chromosome pairs each. The microsatellite distribution pattern was similar in both Progne species, whereas P. cyanoleuca presented a distinct organization. These repetitive DNA sequences were found in the centromeric, pericentromeric, and telomeric regions of the macrochromosomes, as well as in 2 interstitial blocks in the W chromosome. Most microchromosomes had mainly telomeric signals. The Z chromosome displayed 1 hybridization signal in P. tapera but none in the other species. In contrast, the W chromosome showed an accumulation of different microsatellite sequences. The swallow W chromosome is larger than that of most Passeriformes. The observed enlargement in chromosome size might be explained by these high amounts of repetitive sequences. In sum, our data highlight the significant role that microsatellite sequences may play in sex chromosome differentiation.

Correction: Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.)

[This corrects the article DOI: 10.1371/journal.pone.0192862.].

Chromosome-based survey sequencing reveals the genome organization of wild wheat progenitor Triticum dicoccoides.

SummaryWild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome‐based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein‐coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small‐scale evolutionary rearrangements, in addition to providing evidence for the 4AL‐5AL‐7BS translocation in wild emmer wheat. Chromosome‐based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi’s genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a ‘core compartment’ and a ‘disruptive compartment’ which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

Chromosomal distribution of pTa-535, pTa-86, pTa-713, 35S rDNA repetitive sequences in interspecific hexaploid hybrids of common wheat (Triticum aestivum L.) and spelt (Triticum spelta L.).

Fluorescent in situ hybridization (FISH) relies on fluorescent-labeled probes to detect specific DNA sequences in the genome, and it is widely used in cytogenetic analyses. The aim of this study was to determine the karyotype of T. aestivum and T. spelta hybrids and their parental components (three common wheat cultivars and five spelt breeding lines), to identify chromosomal aberrations in the evaluated wheat lines, and to analyze the distribution of polymorphisms of repetitive sequences in the examined hybrids. The FISH procedure was carried out with four DNA clones, pTa-86, pTa-535, pTa-713 and 35S rDNA used as probes. The observed polymorphisms between the investigated lines of common wheat, spelt and their hybrids was relatively low. However, differences were observed in the distribution of repetitive sequences on chromosomes 4A, 6A, 1B and 6B in selected hybrid genomes. The polymorphisms observed in common wheat and spelt hybrids carry valuable information for wheat breeders. The results of our study are also a valuable source of knowledge about genome organization and diversification in common wheat, spelt and their hybrids. The relevant information is essential for common wheat breeders, and it can contribute to breeding programs aimed at biodiversity preservation.

Molecular cytogenetics to characterize mechanisms of gene duplication in pesticide resistance.

Recent advances in molecular cytogenetics empower construction of physical maps to illustrate the precise position of genetic loci on the chromosomes. Such maps provide visible information about the position of DNA sequences, including the distribution of repetitive sequences on the chromosomes. This is an important step toward unraveling the genetic mechanisms implicated in chromosomal aberrations (e.g., gene duplication). In response to stress, such as pesticide selection, duplicated genes provide an immediate adaptive advantage to organisms that overcome unfavorable conditions. Although the significance of gene duplication as one of the important events driving genetic diversity has been reported, the precise mechanisms of gene duplication that contribute to pesticide resistance, especially to herbicides, are elusive. With particular reference to pesticide resistance, we discuss the prospects of application of molecular cytogenetic tools to uncover mechanism(s) of gene duplication, and illustrate hypothetical models that predict the evolutionary basis of gene duplication. The cytogenetic basis of duplicated genes, their stability, as well as the magnitude of selection pressure, can determine the dynamics of the genetic locus (loci) conferring pesticide resistance not only at the population level, but also at the individual level. © 2017 Society of Chemical Industry.

Fluorescence In Situ Hybridization in Oat.

This chapter describes methods to detect gene loci or gene transcripts by fluorescence labeling. Fluorescence in situ hybridization (FISH) can be used to identify the positions of genes or BACs or the distribution of repetitive sequences on metaphase chromosomes as well as the identification of alien chromosomes. It enables the identification of gene loci and active transcription sites in interphase nuclei and also the localization of cellular transcripts. The protocols here deal with the production of DNA and RNA probes, the preparation of oat metaphase spreads and root tissue sections, the subsequent hybridization, post-hybridization washes, and detection by immunofluorescence.

In Situ Hybridization in Rice (Oryza sativa).

Fluorescence in situ hybridization (FISH) is widely used in cytogenetics to determine the localization of DNA sequences on target chromosomes, to provide visible information regarding the physical position of DNA sequences, to determine the abundance and distribution of repetitive sequences that comprise a large proportion of genomes, and to determine the relative chromosome positions of multiple sequences in physical mapping. By mapping on extended chromatin fibers, fiber-FISH can be used to determine the structure and organization of genes or DNA sequences with a high resolution (to a few kilobases). The protocols described here will provide procedures of FISH on metaphase chromosomes and extended chromatin fibers of rice (Oryza sativa). © 2016 by John Wiley & Sons, Inc.

Short tandem repeats in the human, cow, mouse, chicken, and lizard genomes are concentrated in the terminal regions of chromosomes

Repetitive sequences in a genome cause mapping errors, especially in the case of short reads, because of the presence of similar or identical sequences. Distribution of repetitive sequences in a genome must be studied to distinguish between mappable and unmappable regions. Previous studies showed that short tandem repeats (STRs) are clustered in the terminal regions of chromosomes in the human genome. It is an open question whether formation of STRs in the terminal regions of chromosomes occurs only in humans. The present study investigated the distribution of STRs in the human, cow, mouse, chicken, and lizard genomes. In this study, it was shown that STRs were concentrated in the terminal regions of chromosomes not only in the human genome, but also in the mouse, cow, chicken, and lizard genomes. The results suggested the mechanism through which STRs are shared by amniotes in which mammals, birds and lizards are included. Thus, we must be careful with the genomic sequences at chromosome ends of amniotes by using the next generation sequencers.

Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

BackgroundDifferentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats.ResultsChromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo.ConclusionsIn summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1877-6) contains supplementary material, which is available to authorized users.

Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes).

Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.