Heterochromatin: Guardian of the Genome.

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment.

Tandemly repeated genes promote RNAi-mediated heterochromatin formation via an antisilencing factor, Epe1, in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in cooperation with an antisilencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. This "repeat-induced RNAi" depends on the copy number of repeated genes and Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

Functional Diversification of Chromatin on Rapid Evolutionary Timescales.

Repeat-enriched genomic regions evolve rapidly and yet support strictly conserved functions like faithful chromosome transmission and the preservation of genome integrity. The leading resolution to this paradox is that DNA repeat-packaging proteins evolve adaptively to mitigate deleterious changes in DNA repeat copy number, sequence, and organization. Exciting new research has tested this model of coevolution by engineering evolutionary mismatches between adaptively evolving chromatin proteins of one species and the DNA repeats of a close relative. Here, we review these innovative evolution-guided functional analyses. The studies demonstrate that vital, chromatin-mediated cellular processes, including transposon suppression, faithful chromosome transmission, and chromosome retention depend on species-specific versions of chromatin proteins that package species-specific DNA repeats. In many cases, the ever-evolving repeats are selfish genetic elements, raising the possibility that chromatin is a battleground of intragenomic conflict.

Emerging roles of DNA repair factors in the stability of centromeres.

Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.

C-DNA may facilitate homologous DNA pairing.

Recombination-independent homologous pairing represents a prominent yet largely enigmatic feature of chromosome biology. As suggested by studies in the fungus Neurospora crassa, this process may be based on the direct pairing of homologous DNA molecules. Theoretical search for the DNA structures consistent with those genetic results has led to an all-atom model in which the B-DNA conformation of the paired double helices is strongly shifted toward C-DNA. Coincidentally, C-DNA also features a very shallow major groove that could permit initial homologous contacts without atom-atom clashes. The hereby conjectured role of C-DNA in homologous pairing should encourage the efforts to discover its biological functions and may also clarify the mechanism of recombination-independent recognition of DNA homology.

Long-Read DNA Sequencing: Recent Advances and Remaining Challenges.

DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.

Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Novel centromeric repetitive DNA elements reveal karyotype dynamics in polyploid sainfoin (Onobrychis viciifolia).

Polyploidy is a common feature in eukaryotes with one of paramount consequences leading to better environmental adaptation. Heterochromatin is often located at telomeres and centromeres and contains repetitive DNA sequences. Sainfoin (Onobrychis viciifolia) is an important perennial forage legume for sustainable agriculture. However, there are only a few studies on the sainfoin genome and chromosomes. In this study, novel tandem repetitive DNA sequences of the sainfoin genome (OnVi180, OnVi169, OnVi176 and OnVidimer) were characterized using bioinformatics, molecular and cytogenetic approaches. The OnVi180 and OnVi169 elements colocalized within functional centromeres. The OnVi176 and OnVidimer elements were localized in centromeric, subtelomeric and interstitial regions. We constructed a sainfoin karyotype that distinguishes the seven basic chromosome groups. Our study provides the first detailed description of heterochromatin and chromosome structure of sainfoin and proposes an origin of heterozygous ancestral genomes, possibly from the same ancestral diploid species, not necessarily from different species, or for chromosome rearrangements after polyploidy. Overall, we discuss our novel and complementary findings in a polyploid crop with unique and complex chromosomal features.

The Dynamic Interplay Between Ribosomal DNA and Transposable Elements: A Perspective From Genomics and Cytogenetics.

Although both are salient features of genomes, at first glance ribosomal DNAs and transposable elements are genetic elements with not much in common: whereas ribosomal DNAs are mainly viewed as housekeeping genes that uphold all prime genome functions, transposable elements are generally portrayed as selfish and disruptive. These opposing characteristics are also mirrored in other attributes: organization in tandem (ribosomal DNAs) versus organization in a dispersed manner (transposable elements); evolution in a concerted manner (ribosomal DNAs) versus evolution by diversification (transposable elements); and activity that prolongs genomic stability (ribosomal DNAs) versus activity that shortens it (transposable elements). Re-visiting relevant instances in which ribosomal DNA-transposable element interactions have been reported, we note that both repeat types share at least four structural and functional hallmarks: (1) they are repetitive DNAs that shape genomes in evolutionary timescales, (2) they exchange structural motifs and can enter co-evolution processes, (3) they are tightly controlled genomic stress sensors playing key roles in senescence/aging, and (4) they share common epigenetic marks such as DNA methylation and histone modification. Here, we give an overview of the structural, functional, and evolutionary characteristics of both ribosomal DNAs and transposable elements, discuss their roles and interactions, and highlight trends and future directions as we move forward in understanding ribosomal DNA-transposable element associations.

Rpd3 regulates single-copy origins independently of the rDNA array by opposing Fkh1-mediated origin stimulation.

Eukaryotic chromosomes are organized into structural and functional domains with characteristic replication timings, which are thought to contribute to epigenetic programming and genome stability. Differential replication timing results from epigenetic mechanisms that positively and negatively regulate the competition for limiting replication initiation factors. Histone deacetylase Sir2 negatively regulates initiation of the multicopy (∼150) rDNA origins, while Rpd3 histone deacetylase negatively regulates firing of single-copy origins. However, Rpd3's effect on single-copy origins might derive indirectly from a positive function for Rpd3 in rDNA origin firing shifting the competitive balance. Our quantitative experiments support the idea that origins compete for limiting factors; however, our results show that Rpd3's effect on single-copy origin is independent of rDNA copy-number and of Sir2's effects on rDNA origin firing. Whereas RPD3 deletion and SIR2 deletion alter the early S phase dynamics of single-copy and rDNA origin firings in opposite fashion, unexpectedly only RPD3 deletion suppresses overall rDNA origin efficiency across S phase. Increased origin activation in rpd3Δ requires Fkh1/2, suggesting that Rpd3 opposes Fkh1/2-origin stimulation, which involves recruitment of Dbf4-dependent kinase (DDK). Indeed, Fkh1 binding increases at Rpd3-regulated origins in rpd3Δ cells in G1, supporting a mechanism whereby Rpd3 influences initiation timing of single-copy origins directly through modulation of Fkh1-origin binding. Genetic suppression of a DBF4 hypomorphic mutation by RPD3 deletion further supports the conclusion that Rpd3 impedes DDK recruitment by Fkh1, revealing a mechanism of Rpd3 in origin regulation.

Evolution of ancient satellite DNAs in extant alligators and caimans (Crocodylia, Reptilia).

BACKGROUND: Crocodilians are one of the oldest extant vertebrate lineages, exhibiting a combination of evolutionary success and morphological resilience that has persisted throughout the history of life on Earth. This ability to endure over such a long geological time span is of great evolutionary importance. Here, we have utilized the combination of genomic and chromosomal data to identify and compare the full catalogs of satellite DNA families (satDNAs, i.e., the satellitomes) of 5 out of the 8 extant Alligatoridae species. As crocodilian genomes reveal ancestral patterns of evolution, by employing this multispecies data collection, we can investigate and assess how satDNA families evolve over time. RESULTS: Alligators and caimans displayed a small number of satDNA families, ranging from 3 to 13 satDNAs in A. sinensis and C. latirostris, respectively. Together with little variation both within and between species it highlighted long-term conservation of satDNA elements throughout evolution. Furthermore, we traced the origin of the ancestral forms of all satDNAs belonging to the common ancestor of Caimaninae and Alligatorinae. Fluorescence in situ experiments showed distinct hybridization patterns for identical orthologous satDNAs, indicating their dynamic genomic placement. CONCLUSIONS: Alligators and caimans possess one of the smallest satDNA libraries ever reported, comprising only four sets of satDNAs that are shared by all species. Besides, our findings indicated limited intraspecific variation in satellite DNA, suggesting that the majority of new satellite sequences likely evolved from pre-existing ones.

The regulation and potential functions of intronic satellite DNA.

Satellite DNAs are arrays of tandem repeats found in the eukaryotic genome. They are mainly found in pericentromeric heterochromatin and have been believed to be mostly inert, leading satellite DNAs to be erroneously regarded as junk. Recent studies have started to elucidate the function of satellite DNA, yet little is known about the peculiar case where satellite DNA is found within the introns of protein coding genes, resulting in incredibly large introns, a phenomenon termed intron gigantism. Studies in Drosophila demonstrated that satellite DNA-containing introns are transcribed with the gene and require specialized mechanisms to overcome the burdens imposed by the extremely long stretches of repetitive DNA. Whether intron gigantism confers any benefit or serves any functional purpose for cells and/or organisms remains elusive. Here we review our current understanding of intron gigantism: where it is found, the challenges it imposes, how it is regulated and what purpose it may serve.

A classical revival: Human satellite DNAs enter the genomics era.

The classical human satellite DNAs, also referred to as human satellites 1, 2 and 3 (HSat1, HSat2, HSat3, or collectively HSat1-3), occur on most human chromosomes as large, pericentromeric tandem repeat arrays, which together constitute roughly 3% of the human genome (100 megabases, on average). Even though HSat1-3 were among the first human DNA sequences to be isolated and characterized at the dawn of molecular biology, they have remained almost entirely missing from the human genome reference assembly for 20 years, hindering studies of their sequence, regulation, and potential structural roles in the nucleus. Recently, the Telomere-to-Telomere Consortium produced the first truly complete assembly of a human genome, paving the way for new studies of HSat1-3 with modern genomic tools. This review provides an account of the history and current understanding of HSat1-3, with a view towards future studies of their evolution and roles in health and disease.

Mutation protocols share with sexual reproduction the physiological role of producing genetic variation within 'constraints that deconstrain'.

Because the universe of possible DNA sequences is inconceivably vast, organisms have evolved mechanisms for exploring DNA sequence space while substantially reducing the hazard that would otherwise accrue to any process of random, accidental mutation. One such mechanism is meiotic recombination. Although sexual reproduction imposes a seemingly paradoxical 50% cost to fitness, sex evidently prevails because this cost is outweighed by the advantage of equipping offspring with genetic variation to accommodate environmental vicissitudes. The potential adaptive utility of additional mechanisms for producing genetic variation has long been obscured by a presumption that the vast majority of mutations are deleterious. Perhaps surprisingly, the probability for adaptive variation can be increased by several mechanisms that generate mutations abundantly. Such mechanisms, here called 'mutation protocols', implement implicit 'constraints that deconstrain'. Like meiotic recombination, they produce genetic variation in forms that minimize potential for harm while providing a reasonably high probability for benefit. One example is replication slippage of simple sequence repeats (SSRs); this process yields abundant, reversible mutations, typically with small quantitative effect on phenotype. This enables SSRs to function as adjustable 'tuning knobs'. There exists a clear pathway for SSRs to be shaped through indirect selection favouring their implicit tuning-knob protocol. Several other molecular mechanisms comprise probable components of additional mutation protocols. Biologists might plausibly regard such mechanisms of mutation not primarily as sources of deleterious genetic mistakes but also as potentially adaptive processes for 'exploring' DNA sequence space.

The first two whole mitochondrial genomes for the genus Dactylis species: assembly and comparative genomics analysis.

BACKGROUND: Orchardgrass (Dactylis glomerata L.), a perennial forage, has the advantages of rich leaves, high yield, and good quality and is one of the most significant forage for grassland animal husbandry and ecological management in southwest China. Mitochondrial (mt) genome is one of the major genetic systems in plants. Studying the mt genome of the genus Dactylis could provide more genetic information in addition to the nuclear genome project of the genus. RESULTS: In this study, we sequenced and assembled two mitochondrial genomes of Dactylis species of D. glomerata (597, 281 bp) and D. aschersoniana (613, 769 bp), based on a combination of PacBio and Illumina. The gene content in the mitochondrial genome of D. aschersoniana is almost identical to the mitochondrial genome of D. glomerata, which contains 22-23 protein-coding genes (PCGs), 8 ribosomal RNAs (rRNAs) and 30 transfer RNAs (tRNAs), while D. glomerata lacks the gene encoding the Ribosomal protein (rps1) and D. aschersoniana contains one pseudo gene (atp8). Twenty-three introns were found among eight of the 30 protein-coding genes, and introns of three genes (nad 1, nad2, and nad5) were trans-spliced in Dactylis aschersoniana. Further, our mitochondrial genome characteristics investigation of the genus Dactylis included codon usage, sequences repeats, RNA editing and selective pressure. The results showed that a large number of short repetitive sequences existed in the mitochondrial genome of D. aschersoniana, the size variation of two mitochondrial genomes is due largely to the presence of a large number of short repetitive sequences. We also identified 52-53 large fragments that were transferred from the chloroplast genome to the mitochondrial genome, and found that the similarity was more than 70%. ML and BI methods used in phylogenetic analysis revealed that the evolutionary status of the genus Dactylis. CONCLUSIONS: Thus, this study reveals the significant rearrangements in the mt genomes of Pooideae species. The sequenced Dactylis mt genome can provide more genetic information and improve our evolutionary understanding of the mt genomes of gramineous plants.

High-fidelity (repeat) consensus sequences from short reads using combined read clustering and assembly.

BACKGROUND: Despite the many cheap and fast ways to generate genomic data, good and exact genome assembly is still a problem, with especially the repeats being vastly underrepresented and often misassembled. As short reads in low coverage are already sufficient to represent the repeat landscape of any given genome, many read cluster algorithms were brought forward that provide repeat identification and classification. But how can trustworthy, reliable and representative repeat consensuses be derived from unassembled genomes? RESULTS: Here, we combine methods from repeat identification and genome assembly to derive these robust consensuses. We test several use cases, such as (1) consensus building from clustered short reads of non-model genomes, (2) from genome-wide amplification setups, and (3) specific repeat-centred questions, such as the linked vs. unlinked arrangement of ribosomal genes. In all our use cases, the derived consensuses are robust and representative. To evaluate overall performance, we compare our high-fidelity repeat consensuses to RepeatExplorer2-derived contigs and check, if they represent real transposable elements as found in long reads. Our results demonstrate that it is possible to generate useful, reliable and trustworthy consensuses from short reads by a combination from read cluster and genome assembly methods in an automatable way. CONCLUSION: We anticipate that our workflow opens the way towards more efficient and less manual repeat characterization and annotation, benefitting all genome studies, but especially those of non-model organisms.

Bread wheat satellitome: a complex scenario in a huge genome.

In bread wheat (Triticum aestivum L.), chromosome associations during meiosis are extremely regulated and initiate at the telomeres and subtelomeres, which are enriched in satellite DNA (satDNA). We present the study and characterization of the bread wheat satellitome to shed light on the molecular organization of wheat subtelomeres. Our results revealed that the 2.53% of bread wheat genome is composed by satDNA and subtelomeres are particularly enriched in such DNA sequences. Thirty-four satellite DNA (21 for the first time in this work) have been identified, analyzed and cytogenetically validated. Many of the satDNAs were specifically found at particular subtelomeric chromosome regions revealing the asymmetry in subtelomere organisation among the wheat subgenomes, which might play a role in proper homologous recognition and pairing during meiosis. An integrated physical map of the wheat satellitome was also constructed. To the best of our knowledge, our results show that the combination of both cytogenetics and genome research allowed the first comprehensive analysis of the wheat satellitome, shedding light on the complex wheat genome organization, especially on the polymorphic nature of subtelomeres and their putative implication in chromosome recognition and pairing during meiosis.

Revealing the Satellite DNA Content in Ancistrus sp. (Siluriformes: Loricariidae) by Genomic and Bioinformatic Analysis.

INTRODUCTION: Eukaryotic genomes are composed of simple, repetitive sequences, including satellite DNAs (satDNA), which are noncoding sequences arranged in tandem arrays. These sequences play a crucial role in genomic functions and innovations, influencing processes such as the maintenance of nuclear material, the formation of heterochromatin and the differentiation of sex chromosomes. In this genomic era, advances in next-generation sequencing and bioinformatics tools have facilitated the exhaustive cataloging of repetitive elements in genomes, particularly in non-model species. This study focuses on the satDNA content of Ancistrus sp., a diverse species of fish from the Loricariidae family. The genus Ancistrus shows significant karyotypic evolution, with extensive variability from the ancestral diploid number. METHODS: By means of bioinformatic approaches, 40 satDNA families in Ancistrus sp., constituting 5.19% of the genome were identified. Analysis of the abundance and divergence landscape revealed diverse profiles, indicating recent amplification and homogenization of these satDNA sequences. RESULTS: The most abundant satellite, AnSat1-142, constitutes 2.1% of the genome, while the least abundant, AnSat40-52, represents 0.0034%. The length of the monomer repeat varies from 16 to 142 base pairs, with an average length of 61 bp. These results contribute to understanding the genomic dynamics and evolution of satDNAs in Ancistrus sp. CONCLUSION: The study underscores the variability of satDNAs between fish species and provides valuable information on chromosome organization and the evolution of repetitive elements in non-model organisms.

First Karyotypic Insights into Potamotrygon schroederi Fernández-Yépez, 1958: Association of Different Classes of Repetitive DNA.

INTRODUCTION: Currently, there are 38 valid species of freshwater stingrays, and these belong to the subfamily Potamotrygoninae. However, cytogenetic information about this group is limited, with studies mainly using classical techniques, Giemsa, and C-banding. METHODS: In this study, we used classical and molecular cytogenetic techniques - mapping of 18S and 5S rDNA and simple sequence repeats (SSRs) - in order to investigate the karyotypic composition of Potamotrygon schroederi and reveal the karyoevolutionary trends of this group. RESULTS: The species presented 2n = 66 chromosomes with 18m + 12sm + 16st + 20a, heterochromatic blocks distributed in the centromeric regions of all the chromosomes, and terminal blocks in the q arm of pairs 2 and 3. Mapping of 18S rDNA regions revealed multiple clusters on pairs 2 and 7 and a homolog of pair 24. The 5S rDNA region was found in the pericentromeric portion of the subtelocentric pair 16. Furthermore, dinucleotide SSRs sequences were found in the centromeric and terminal regions of different chromosomal pairs, with preferential accumulation in pair 17. In addition, we identified conspicuous blocks of (GATA)n and (GACA)n sequences colocalized with the 5S rDNA (pair 16). CONCLUSION: In general, this study corroborates the general trend of a reduction in 2n in the species of Potamotrygoninae subfamily. Moreover, we found that the location of rDNA regions is very similar among Potamotrygon species, and the SSRs accumulation in the second subtelocentric pair (17) seems to be a common trait in this genus.

In silico Characterization of Satellitomes and Cross-Amplification of Putative satDNAs in Two Species of the Hypostomus ancistroides Complex (Siluriformes, Loricariidae).

INTRODUCTION: The mapping of the satellite DNA on chromosomes is vital to understanding the distribution and evolution of repetitions in the genome since these chromosomal studies have shown the origin, evolutionary mode, and function of repetitive sequences. This study aimed to prospect the satellitome and determine its location in the genome of two cryptic species of Hypostomus, H. aff. ancistroides and H. ancistroides, with and without XX/XY sexual chromosome system. METHODS: Mitotic chromosomes and DNA extraction were obtained according to protocols. After the whole genome sequencing, the satDNAs were retrieved, amplified, and hybridized in chromosome preparations for male and female individuals. RESULTS: We found 30 satellite families (47 variants, two superfamilies) in H. ancistroides and 38 satellite families (45 variants, four superfamilies) in H. aff. ancistroides. The sequences varied from 14 bp to 2,662 bp in H. ancistroides and from 14 bp to 2,918 bp in H. aff. ancistroides. We did not observe any tandem repeats that were exclusive to each of the libraries; however, many sequences showed very different abundances and copy numbers between the libraries. Four satDNAs did not hybridize on the chromosomes of either species. Conversely, one satDNA hybridized in both species, HxySat1-80. However, the phenotypes found varied among species, populations, and in the same individual. There was no sign of HanSat3-464 and HanSat11-335 in any individuals of H. aff. ancistroides, but markings were in the chromosomes of H. ancistroides. HxySat12-1127 and HxySat8-52, on the other hand, were only hybridized in H. aff. ancistroides, while H. ancistroides had a negative sign. No hybridization of satDNAs was found in the X and Y sex chromosomes as they were mostly composed of euchromatin. CONCLUSION: We distinguish H. aff. ancistroides as genetically different from H. ancistroides, recognizing that such characteristics go far beyond morphological, karyotypic, and molecular data. Our data support the differential abundance and location of satellite DNAs and confirm that many organisms, including fish, have repetitive sequences that validate the library hypothesis. All found and validated satDNAs and the characterization of the satellitomes of the two species represent important contributions to cytogenomic studies of the genus Hypostomus.