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Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
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Citosina , Metilación de ADN , ADN Ribosómico , Silenciador del Gen , Citosina/metabolismo , ADN Ribosómico/genética , Variaciones en el Número de Copia de ADN , Transcripción Genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Cromosomas de las Plantas/genéticaRESUMEN
While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.
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Cardamine , Silenciador del Gen , Filogenia , Poliploidía , ARN Ribosómico 5S , ARN Ribosómico 5S/genética , Cardamine/genética , Genoma de Planta/genética , ADN Ribosómico/genética , Hibridación Fluorescente in Situ , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
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Elementos Transponibles de ADN , Genómica , ADN Ribosómico , Metilación de ADN , Análisis Citogenético , Evolución MolecularRESUMEN
BACKGROUND AND AIMS: Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2nâ =â 5xâ =â 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. METHODS: We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. KEY RESULTS: In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. CONCLUSIONS: We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.
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Histonas , Meiosis , Histonas/genética , Fosforilación , Hibridación Fluorescente in Situ , Cromosomas , Epigénesis GenéticaRESUMEN
Accumulation and selection of nucleotides is one of the most challenging problems surrounding the origin of the first RNA molecules on our planet. In the current work we propose that guanosine 3',5' cyclic monophosphate could selectively crystallize upon evaporation of an acidic prebiotic pool containing various other nucleotides. The conditions of the evaporative crystallization are fully compatible with the subsequent acid catalyzed polymerization of this cyclic nucleotide reported in earlier studies and may be relevant in a broad range of possible prebiotic environments. Albeit cytidine 3',5' cyclic monophosphate has the ability to selectively accumulate under the same conditions, its crystal structure is not likely to support polymer formation.
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The supernumerary mostly dispensable B chromosomes are nuclear components of about 15% of eukaryotic phyla. For a long time, B chromosomes have been studied, generating an enormous bulk of knowledge, diluted in the vastness of the scientific literature. In order to provide better access to this information, we created B-chrom ( www.bchrom.csic.es ), an online database with comprehensive information on Bs for plants, animals, and fungi. It was released in 2017 and first updated in 2021, by adding 334 entries and 123 new species. Currently, the resource provides information for 2951 species coming from 3292 sources. During this time, the usefulness of this database has been proven by the number of visits (more than 207,000 since its release) and by the scientific community, having been cited in more than 60 publications until present. This chapter explains the database composition and tips on how to use it.
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Cromo , Eucariontes , Animales , Bases de Datos Factuales , Células Eucariotas , CromosomasRESUMEN
This paper presents the latest update to the Plant rDNA database (Release 4.0), a valuable resource for researchers in the field of plant cytogenetics. The database provides information on the number, position, and arrangement of ribosomal DNA loci in plants, including angiosperms, gymnosperms, bryophytes, and pteridophytes. The new release includes new data for 820 species coming from additional 173 papers. In the updated version of the Plant rDNA database, 4948 entries comprising 2760 organisms can be found. A brief guide on how to navigate the database and obtain the desired information is also provided. The regular updating of the database is important for ensuring the information it contains is accurate, up-to-date, and useful for the research community. The Plant rDNA database continues to be beneficial for phylogenetic and cytogenetic studies in a wide range of taxa including angiosperms, gymnosperms, and early diverging groups, such as bryophytes and lycophytes.
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Fuentes de Información , Magnoliopsida , ADN Ribosómico/genética , Filogenia , Ribosomas , ADN de Plantas/genética , Análisis CitogenéticoRESUMEN
The classical model of concerted evolution states that hundreds to thousands of ribosomal DNA (rDNA) units undergo homogenization, making the multiple copies of the individual units more uniform across the genome than would be expected given mutation frequencies and gene redundancy. While the universality of this over 50-year-old model has been confirmed in a range of organisms, advanced high throughput sequencing techniques have also revealed that rDNA homogenization in many organisms is partial and, in rare cases, even apparently failing. The potential underpinning processes leading to unexpected intragenomic variation have been discussed in a number of studies, but a comprehensive understanding remains to be determined. In this work, we summarize information on variation or polymorphisms in rDNAs across a wide range of taxa amongst animals, fungi, plants, and protists. We discuss the definition and description of concerted evolution and describe whether incomplete concerted evolution of rDNAs predominantly affects coding or non-coding regions of rDNA units and if it leads to the formation of pseudogenes or not. We also discuss the factors contributing to rDNA variation, such as interspecific hybridization, meiotic cycles, rDNA expression status, genome size, and the activity of effector genes involved in genetic recombination, epigenetic modifications, and DNA editing. Finally, we argue that a combination of approaches is needed to target genetic and epigenetic phenomena influencing incomplete concerted evolution, to give a comprehensive understanding of the evolution and functional consequences of intragenomic variation in rDNA.
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Variación Genética , Polimorfismo Genético , Animales , ADN Ribosómico/genética , Mutación , Hongos/genética , Evolución Molecular , FilogeniaRESUMEN
The ribosomal RNA genes (rDNA) are universal genome components with a housekeeping function, given the crucial role of ribosomal RNA in the synthesis of ribosomes and thus for life-on-Earth. Therefore, their genomic organization is of considerable interest for biologists, in general. Ribosomal RNA genes have also been largely used to establish phylogenetic relationships, and to identify allopolyploid or homoploid hybridization.Here, we demonstrate how high-throughput sequencing data, through graph clustering implemented in RepeatExplorer2 pipeline ( https://repeatexplorer-elixir.cerit-sc.cz/galaxy/ ), can be helpful to decipher the genomic organization of 5S rRNA genes. We show that the linear shapes of cluster graphs are reminiscent to the linked organization of 5S and 35S rDNA (L-type arrangement) while the circular graphs correspond to their separate arrangement (S-type). We further present a simplified protocol based on the paper by (Garcia et al., Front Plant Sci 11:41, 2020) about the use of graph clustering of 5S rDNA homoeologs (S-type) to identify hybridization events in the species history. We found that the graph complexity (i.e., graph circularity in this case) is related to ploidy and genome complexity, with diploids typically showing circular-shaped graphs while allopolyploids and other interspecific hybrids display more complex graphs, with usually two or more interconnected loops representing intergenic spacers. When a three-genomic comparative clustering analysis from a given hybrid (homoploid/allopolyploid) and its putative progenitor species (diploids) is performed, it is possible to identify the corresponding homoeologous 5S rRNA gene families, and to elucidate the contribution of each putative parental genome to the 5S rDNA pool of the hybrid. Thus, the analysis of 5S rDNA cluster graphs by RepeatExplorer, together with information coming from other sources (e.g., morphology, cytogenetics) is a complementary approach for the determination of allopolyploid or homoploid hybridization and even ancient introgression events.
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Genómica , ARN Ribosómico 5S , ADN Ribosómico/genética , Filogenia , ARN Ribosómico 5S/genética , Genes de ARNrRESUMEN
RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.
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Citidina , ARN , ARN/metabolismo , Citidina/genética , Citidina/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Cromatina , AcetilaciónRESUMEN
Nucleolar dominance (ND) is selective epigenetic silencing of 35-48S rDNA loci. In allopolyploids, it is frequently manifested at the cytogenetic level by the inactivation of nucleolar organiser region(s) (NORs) inherited from one or several evolutionary ancestors. Grasses are ecologically and economically one of the most important land plant groups, which have frequently evolved through hybridisation and polyploidisation events. Here we review common and unique features of ND phenomena in this monocot family from cytogenetic, molecular, and genomic perspectives. We highlight recent advances achieved by using an allotetraploid model grass, Brachypodium hybridum, where ND commonly occurs at a population level, and we cover modern genomic approaches that decipher structural features of core arrays of NORs.
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Nucléolo Celular , Región Organizadora del Nucléolo , Genes de ARNr , ADN Ribosómico/genética , Nucléolo Celular/genética , Poaceae/genéticaRESUMEN
Simple telomeric repeats composed of six to seven iterating nucleotide units are important sequences typically found at the ends of chromosomes. Here we analyzed their abundance and homogeneity in 42 gymnosperm (29 newly sequenced), 29 angiosperm (one newly sequenced), and eight bryophytes using bioinformatics, conventional cytogenetic and molecular biology approaches to explore their diversity across land plants. We found more than 10 000-fold variation in the amounts of telomeric repeats among the investigated taxa. Repeat abundance was positively correlated with increasing intragenomic sequence heterogeneity and occurrence at non-telomeric positions, but there was no correlation with genome size. The highest abundance/heterogeneity was found in the gymnosperm genus Cycas (Cycadaceae), in which megabase-sized blocks of telomeric repeats (i.e., billions of copies) were identified. Fluorescent in situ hybridization experiments using variant-specific probes revealed canonical Arabidopsis-type telomeric TTTAGGG repeats at chromosome ends, while pericentromeric blocks comprised at least four major telomeric variants with decreasing abundance: TTTAGGG>TTCAGGG >TTTAAGG>TTCAAGG. Such a diversity of repeats was not found in the sister cycad family Zamiaceae or in any other species analyzed. Using immunocytochemistry, we showed that the pericentromeric blocks of telomeric repeats overlapped with histone H3 serine 10 phosphorylation signals. We show that species of Cycas have amplified their telomeric repeats in centromeric and telomeric positions on telocentric chromosomes to extraordinary high levels. The ancestral chromosome number reconstruction suggests their occurrence is unlikely to be the product of ancient Robertsonian chromosome fusions. We speculate as to how the observed chromosome dynamics may be associated with the diversification of cycads.
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Cycadopsida , Magnoliopsida , Hibridación Fluorescente in Situ , Cycadopsida/genética , Telómero/genética , Centrómero/genética , Magnoliopsida/genéticaRESUMEN
[This corrects the article DOI: 10.3389/fpls.2021.768347.].
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During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised the possibility of horizontal transfer of a eudicot alien ribosomal DNA (rDNA) into the Erythronium genome. In this work we aimed to support this hypothesis by carrying out genomic, molecular, and cytogenetic analyses. Genome skimming coupled by PacBio HiFi sequencing of a bacterial artificial chromosome clone derived from flow-sorted nuclei was used to characterise the alien 45S rDNA. Integration of alien rDNA in the recipient genome was further proved by Southern blotting and fluorescence in situ hybridization using specific probes. Alien rDNA, nested among Potentilla species in phylogenetic analysis, likely entered the Erythronium lineage in the common ancestor of E. dens-canis and E. caucasicum. Transferred eudicot-type rDNA preserved its tandemly arrayed feature on a single chromosome and was found to be transcribed in the monocot host, albeit much less efficiently than the native counterpart. This study adds a new example to the rarely documented nuclear-to-nuclear jumps of DNA between eudicots and monocots while holding the scientific community continually in suspense about the mode of DNA transfer.
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Liliaceae , Potentilla , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Hibridación Fluorescente in Situ , Filogenia , Potentilla/genéticaRESUMEN
Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi-omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus-specific analyses of transcription activity and methylation status from RNA- and bisulfite-sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue-specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.
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Pan , Triticum , ADN Ribosómico/genética , Poliploidía , ARN Ribosómico/genética , Triticum/genéticaRESUMEN
Nucleolar dominance (ND) is an epigenetic, developmentally regulated phenomenon that describes the selective inactivation of 35S rDNA loci derived from one progenitor of a hybrid or allopolyploid. The presence of ND was documented in an allotetraploid grass, Brachypodium hybridum (genome composition DDSS), which is a polyphyletic species that arose from crosses between two putative ancestors that resembled the modern B. distachyon (DD) and B. stacei (SS). In this work, we investigated the developmental stability of ND in B. hybridum genotype 3-7-2 and compared it with the reference genotype ABR113. We addressed the question of whether the ND is established in generative tissues such as pollen mother cells (PMC). We examined condensation of rDNA chromatin by fluorescence in situ hybridization employing state-of-art confocal microscopy. The transcription of rDNA homeologs was determined by reverse-transcription cleaved amplified polymorphic sequence analysis. In ABR113, the ND was stable in all tissues analyzed (primary and adventitious root, leaf, and spikes). In contrast, the 3-7-2 individuals showed a strong upregulation of the S-genome units in adventitious roots but not in other tissues. Microscopic analysis of the 3-7-2 PMCs revealed extensive decondensation of the D-genome loci and their association with the nucleolus in meiosis. As opposed, the S-genome loci were always highly condensed and localized outside the nucleolus. These results indicate that genotype-specific loss of ND in B. hybridum occurs probably after fertilization during developmental processes. This finding supports our view that B. hybridum is an attractive model to study ND in grasses.
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Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a "repeatomic fingerprint," and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa, at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete repeatomes with those from ribosomal DNA clusters only, because they represent one of the most widely used barcoding markers. Our results demonstrated that repeatome data contained a clear phylogenetic signal supporting the current subfamilial classification within Rosaceae. Accordingly, the well-accepted major evolutionary lineages within Fragaria were distinguished, and hybrids showed intermediate positions between parental species in data sets retrieved from both complete repeatomes and rDNA clusters. Within the taxonomically more complicated and particularly frequently hybridizing genus Rosa, we detected rather weak phylogenetic signals but surprisingly found a geographic pattern at a population scale. In sum, our method revealed promising results at larger taxonomic scales as well as within taxa with manageable levels of reticulation, but success remained rather taxon specific. Since repeatomes can be technically easy and comparably inexpensively retrieved even from samples of rather poor DNA quality, our phylogenomic method serves as a valuable alternative when high-quality genomes are unavailable, for example, in the case of old museum specimens.
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The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3',5'-cyclic guanosine monophosphate (3',5'-cGMP) has been proposed as a candidate to create oligonucleotides of 15-20â units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pHâ 9.