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.

• Keywords
• 35S rDNA copy number variations, CCGs and CHHs, CWGs, Tragopogon porrifolius ssp. porrifolius, bidirectional nucleolar dominance, methylation dynamics of CGs, transcriptional silencing/activation,
• MeSH
• Chromosomes, Plant genetics   MeSH
• Cytosine * metabolism   MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic MeSH
• DNA Methylation * MeSH
• Gene Expression Regulation, Plant MeSH
• DNA, Ribosomal * genetics   MeSH
• Gene Silencing * MeSH
• DNA Copy Number Variations MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytosine * MeSH
• DNA, Ribosomal * MeSH

BACKGROUND AND AIMS: Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 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.

• Keywords
• Rosa genus, 18S ribosomal DNA, canina meiosis, dogroses, euchromatin and heterochromatin, fluorescence in situ hybridization, histone H3 phosphorylation, immunostaining, non-disjunction,
• MeSH
• Chromosomes MeSH
• Epigenesis, Genetic MeSH
• Phosphorylation MeSH
• Histones * genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Meiosis * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Histones * MeSH

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.

• Keywords
• concerted evolution, genome size, genome stability, homogenization, housekeeping genes, long-read sequencing, molecular cytogenetics, recombination, repetitive DNA, ribosomal DNA, transposable elements, transposition,
• MeSH
• Cytogenetic Analysis MeSH
• Genomics * MeSH
• DNA Methylation MeSH
• Evolution, Molecular MeSH
• DNA, Ribosomal MeSH
• DNA Transposable Elements * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal MeSH
• DNA Transposable Elements * MeSH

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.

• Keywords
• Chromosome, Comparative cytogenetics, Data mining, In situ hybridization, Karyotype, L-type arrangement, Ribosomal DNA, S-type arrangement,
• MeSH
• Cytogenetic Analysis MeSH
• DNA, Plant genetics   MeSH
• Phylogeny MeSH
• Information Sources * MeSH
• Magnoliopsida * MeSH
• DNA, Ribosomal genetics   MeSH
• Ribosomes MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Ribosomal MeSH

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.

• MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Fungi genetics   MeSH
• Evolution, Molecular MeSH
• Mutation MeSH
• Polymorphism, Genetic * MeSH
• DNA, Ribosomal genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA, Ribosomal MeSH

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.

• Keywords
• 5S ribosomal RNA genes, Allopolyploid hybridization, Genomic analysis, Graph clustering, Homoploid hybridization, Introgression, Repeatome,
• MeSH
• Phylogeny MeSH
• Genomics * MeSH
• Genes, rRNA MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 5S * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 5S * MeSH

Genes for major ribosomal RNAs (rDNA) are present in multiple copies mainly organized in tandem arrays. The number and position of rDNA loci can change dynamically and their repatterning is presumably driven by other repetitive sequences. We explored a peculiar rDNA organization in several representatives of Lepidoptera with either extremely large or numerous rDNA clusters. We combined molecular cytogenetics with analyses of second- and third-generation sequencing data to show that rDNA spreads as a transcription unit and reveal association between rDNA and various repeats. Furthermore, we performed comparative long read analyses among the species with derived rDNA distribution and moths with a single rDNA locus, which is considered ancestral. Our results suggest that satellite arrays, rather than mobile elements, facilitate homology-mediated spread of rDNA via either integration of extrachromosomal rDNA circles or ectopic recombination. The latter arguably better explains preferential spread of rDNA into terminal regions of lepidopteran chromosomes as efficiency of ectopic recombination depends on the proximity of homologous sequences to telomeres.

• Keywords
• Lepidoptera, major ribosomal RNA genes, mobile elements, satellite,
• MeSH
• Chromosomes MeSH
• Moths * genetics   MeSH
• Repetitive Sequences, Nucleic Acid * MeSH
• DNA, Ribosomal genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss - from a personal view - the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on "Molecular organization, evolution, and function of ribosomal DNA."

• Keywords
• epigenetics, hybridization, molecular evolution, nucleolar dominance, polyploidy, rDNA research history, rRNA precursor, rRNA processing,
• Publication type
• Journal Article MeSH
• Review MeSH

We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively.The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists. .

• Keywords
• Ag-NOR, B chromosome, animals, database, karyotype, nucleolar organizer regions, rDNA, rRNA genes, ribosomal DNA, sex chromosome,
• MeSH
• Databases, Genetic MeSH
• Species Specificity MeSH
• Karyotype MeSH
• Karyotyping MeSH
• Evolution, Molecular MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• RNA, Ribosomal genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 5S MeSH
• RNA, Ribosomal MeSH
• RNA, ribosomal, 45S MeSH Browser

The genus Trifolium L. is characterized by basic chromosome numbers 8, 7, 6, and 5. We conducted a genus-wide study of ribosomal DNA (rDNA) structure variability in diploids and polyploids to gain insight into evolutionary history. We used fluorescent in situ hybridization to newly investigate rDNA variation by number and position in 30 Trifolium species. Evolutionary history among species was examined using 85 available sequences of internal transcribed spacer 1 (ITS1) of 35S rDNA. In diploid species with ancestral basic chromosome number (x = 8), one pair of 5S and 26S rDNA in separate or adjacent positions on a pair of chromosomes was prevalent. Genomes of species with reduced basic chromosome numbers were characterized by increased number of signals determined on one pair of chromosomes or all chromosomes. Increased number of signals was observed also in diploids Trifolium alpestre and Trifolium microcephalum and in polyploids. Sequence alignment revealed ITS1 sequences with mostly single nucleotide polymorphisms, and ITS1 diversity was greater in diploids with reduced basic chromosome numbers compared to diploids with ancestral basic chromosome number (x = 8) and polyploids. Our results suggest the presence of one 5S rDNA site and one 26S rDNA site as an ancestral state.

• Keywords
• 26S rDNA, 5S rDNA, clover, fluorescent in situ hybridization, genome structure, nucleotide polymorphism,
• Publication type
• Journal Article MeSH