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

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.

• Keywords
• Cycadaceae, centromeres, chromosome rearrangements, epigenetics, genome evolution, gymnosperms, telomeres,
• MeSH
• Centromere genetics   MeSH
• Cycadopsida * genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Magnoliopsida * genetics   MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5V569E and MAP4K5V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.

• Keywords
• Cancer, RNA editing, mass spectrometry, proteogenomics, splicing,
• MeSH
• Alternative Splicing * MeSH
• DNA genetics   MeSH
• RNA Editing MeSH
• Exons * MeSH
• Isoenzymes MeSH
• Humans MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• RNA genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Isoenzymes MeSH
• MAP4K5 protein, human MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• RNA 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

Nucleolar dominance (ND) consists of the reversible silencing of 35S/45S rDNA loci inherited from one of the ancestors of an allopolyploid. The molecular mechanisms by which one ancestral rDNA set is selected for silencing remain unclear. We applied a combination of molecular (Southern blot hybridization and reverse-transcription cleaved amplified polymorphic sequence analysis), genomic (analysis of variants) and cytogenetic (fluorescence in situ hybridization) approaches to study the structure, expression and epigenetic landscape of 35S rDNA in an allotetraploid grass that exhibits ND, Brachypodium hybridum (genome composition DDSS), and its putative progenitors, Brachypodium distachyon (DD) and Brachypodium stacei (SS). In progenitor genomes, B. stacei showed a higher intragenomic heterogeneity of rDNA compared with B. distachyon. In all studied accessions of B. hybridum, there was a reduction in the copy number of S homoeologues, which was accompanied by their inactive transcriptional status. The involvement of DNA methylation in CG and CHG contexts in the silencing of the S-genome rDNA loci was revealed. In the B. hybridum allotetraploid, ND is stabilized towards the D-genome units, irrespective of the polyphyletic origin of the species, and does not seem to be influenced by homoeologous 35S rDNA ratios and developmental stage.

• Keywords
• Brachypodium hybridum, 35S rDNA evolution, 35S rRNA gene expression, allopolyploidy, nucleolar dominance,
• MeSH
• Brachypodium genetics   metabolism   MeSH
• Chromosomes, Plant genetics   MeSH
• Genetic Loci genetics   MeSH
• Genome, Plant genetics   MeSH
• Genes, rRNA genetics   MeSH
• DNA Methylation genetics   MeSH
• Evolution, Molecular MeSH
• Polymorphism, Genetic genetics   MeSH
• Genes, Plant genetics   MeSH
• Blotting, Southern MeSH
• Tetraploidy * MeSH
• DNA Copy Number Variations genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.

• Keywords
• CpG depletion, SARS-CoV-2, coronavirus, cytosine deamination, evolution, mutation bias,
• MeSH
• Betacoronavirus classification   genetics   isolation & purification   MeSH
• Chiroptera virology   MeSH
• CpG Islands MeSH
• Cytosine metabolism   MeSH
• Phylogeny MeSH
• Spike Glycoprotein, Coronavirus genetics   MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Evolution, Molecular * MeSH
• SARS-CoV-2 MeSH
• Base Sequence MeSH
• Uracil metabolism   MeSH
• Severe acute respiratory syndrome-related coronavirus classification   genetics   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytosine MeSH
• Spike Glycoprotein, Coronavirus MeSH
• Uracil MeSH

INTRODUCTION: Ribosomal DNA (rDNA) loci have been widely used for identification of allopolyploids and hybrids, although few of these studies employed high-throughput sequencing data. Here we use graph clustering implemented in the RepeatExplorer (RE) pipeline to analyze homoeologous 5S rDNA arrays at the genomic level searching for hybridogenic origin of species. Data were obtained from more than 80 plant species, including several well-defined allopolyploids and homoploid hybrids of different evolutionary ages and from widely dispersed taxonomic groups. RESULTS: (i) Diploids show simple circular-shaped graphs of their 5S rDNA clusters. In contrast, most allopolyploids and other interspecific hybrids exhibit more complex graphs composed of two or more interconnected loops representing intergenic spacers (IGS). (ii) There was a relationship between graph complexity and locus numbers. (iii) The sequences and lengths of the 5S rDNA units reconstituted in silico from k-mers were congruent with those experimentally determined. (iv) Three-genomic comparative cluster analysis of reads from allopolyploids and progenitor diploids allowed identification of homoeologous 5S rRNA gene families even in relatively ancient (c. 1 Myr) Gossypium and Brachypodium allopolyploids which already exhibit uniparental partial loss of rDNA repeats. (v) Finally, species harboring introgressed genomes exhibit exceptionally complex graph structures. CONCLUSION: We found that the cluster graph shapes and graph parameters (k-mer coverage scores and connected component index) well-reflect the organization and intragenomic homogeneity of 5S rDNA repeats. We propose that the analysis of 5S rDNA cluster graphs computed by the RE pipeline together with the cytogenetic analysis might be a reliable approach for the determination of the hybrid or allopolyploid plant species parentage and may also be useful for detecting historical introgression events.

• Keywords
• 5S rRNA genes, allopolyploidy, evolution, graph structure clustering, high-throughput sequencing, hybridization, repeatome,
• Publication type
• Journal Article MeSH

Introduction: In plants, the multicopy genes encoding ribosomal RNA (rDNA) typically exhibit heterochromatic features and high level of DNA methylation. Here, we explored rDNA methylation in early diverging land plants from Bryophyta (15 species, 14 families) and Marchantiophyta (4 species, 4 families). DNA methylation was investigated by methylation-sensitive Southern blot hybridization in all species. We also carried out whole genomic bisulfite sequencing in Polytrichum formosum (Polytrichaceae) and Dicranum scoparium (Dicranaceae) and used available model plant methyloms (Physcomitrella patents and Marchantia polymorpha) to determine rDNA unit-wide methylation patterns. Chromatin structure was analyzed using fluorescence in situ hybridization (FISH) and immunoprecipitation (CHIP) assays. Results: In contrast to seed plants, bryophyte rDNAs were efficiently digested with methylation-sensitive enzymes indicating no or low levels of CG and CHG methylation in these loci. The rDNA methylom analyses revealed variation between species ranging from negligible (<3%, P. formosum, P. patens) to moderate (7 and 17% in M. polymorpha and D. scoparium, respectively) methylation levels. There were no differences between coding and noncoding parts of rDNA units and between gametophyte and sporophyte tissues. However, major satellite repeat and transposable elements were heavily methylated in P. formosum and D. scoparium. In P. formosum rDNA, the euchromatic H3K4m3 and heterochromatic H3K9m2 histone marks were nearly balanced contrasting the angiosperms data where H3K9m2 typically dominates rDNA chromatin. In moss interphase nuclei, rDNA was localized at the nucleolar periphery and its condensation level was high. Conclusions: Unlike seed plants, the rRNA genes seem to escape global methylation machinery in bryophytes. Distinct epigenetic features may be related to rDNA expression and the physiology of these early diverging plants that exist in haploid state for most of their life cycles.

• Keywords
• bryophytes, cytosine methylation, epigenetics, genome evolution, histone marks, rDNA,
• Publication type
• Journal Article MeSH

INTRODUCTION: Gnetophytes, comprising the genera Ephedra, Gnetum and Welwitschia, are an understudied, enigmatic lineage of gymnosperms with a controversial phylogenetic relationship to other seed plants. Here we examined the organization of ribosomal DNA (rDNA) across representative species. METHODS: We applied high-throughput sequencing approaches to isolate and reconstruct rDNA units and to determine their intragenomic homogeneity. In addition, fluorescent in situ hybridization and Southern blot hybridization techniques were used to reveal the chromosome and genomic organization of rDNA. KEY RESULTS: The 5S and 35S rRNA genes were separate (S-type) in Gnetum montanum, Gnetum gnemon and Welwitschia mirabilis and linked (L-type) in Ephedra altissima. There was considerable variability in 5S rDNA abundance, ranging from as few as ~4000 (W. mirabilis) to >100 000 (G. montanum) copies. A similar large variation was also observed in 5S rDNA locus numbers (two to 16 sites per diploid cell). 5S rRNA pseudogenes were interspersed between functional genes forming a single unit in E. altissima and G. montanum. Their copy number was comparable or even higher than that of functional 5S rRNA genes. In E. altissima internal transcribed spacers of 35S rDNA were long and intrinsically repetitive while in G. montanum and W. mirabilis they were short without the subrepeats. CONCLUSIONS: Gnetophytes are distinct from other gymnosperms and angiosperms as they display surprisingly large variability in rDNA organization and rDNA copy and locus numbers between genera, with no relationship between copy numbers and genome sizes apparent. Concerted evolution of 5S rDNA units seems to have led to the amplification of 5S pseudogenes in both G. montanum and E. altissima. Evolutionary patterns of rDNA show both gymnosperm and angiosperm features underlining the diversity of the group.

• Keywords
• Gnetophytes, chromosome evolution, concerted evolution, high-throughput sequencing, intragenomic diversity, pseudogenes, rDNA organization,
• MeSH
• Cycadopsida * MeSH
• Phylogeny MeSH
• In Situ Hybridization, Fluorescence MeSH
• Evolution, Molecular MeSH
• DNA, Ribosomal MeSH
• DNA Copy Number Variations MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH