BACKGROUND: Polyploidisation often results in genome rearrangements that may involve changes in both the single-copy sequences and the repetitive genome fraction. In this study, we performed a comprehensive comparative analysis of repetitive DNA, with a particular focus on ribosomal DNA (rDNA), in Brachypodium hybridum (2n = 4x = 30, subgenome composition DDSS), an allotetraploid resulting from a natural cross between two diploid species that resemble the modern B. distachyon (2n = 10; DD) and B. stacei (2n = 20; SS). Taking advantage of the recurrent origin of B. hybridum, we investigated two genotypes, Bhyb26 and ABR113, differing markedly in their evolutionary age (1.4 and 0.14 Mya, respectively) and which resulted from opposite cross directions. To identify the origin of rDNA loci we employed cytogenetic and molecular methods (FISH, gCAPS and Southern hybridisation), phylogenetic and genomic approaches. RESULTS: Unlike the general maintenance of doubled gene dosage in B. hybridum, the rRNA genes showed a remarkable tendency towards diploidisation at both locus and unit levels. While the partial elimination of 35S rDNA units occurred in the younger ABR113 lineage, unidirectional elimination of the entire locus was observed in the older Bhyb26 lineage. Additionally, a novel 5S rDNA family was amplified in Bhyb26 replacing the parental units. The 35S and 5S rDNA units were preferentially eliminated from the S- and D-subgenome, respectively. Thus, in the more ancient B. hybridum lineage, Bhyb26, 5S and 35S rRNA genes are likely expressed from different subgenomes, highlighting the complexity of polyploid regulatory networks. CONCLUSION: Comparative analyses between two B. hybridum lineages of distinct evolutionary ages revealed that although the recent lineage ABR113 exhibited an additive pattern of rDNA loci distribution, the ancient lineage Bhyb26 demonstrated a pronounced tendency toward diploidisation manifested by the reduction in the number of both 35S and 5S loci. In conclusion, the age of the allopolyploid appears to be a decisive factor in rDNA turnover in B. hybridum.

• Klíčová slova
• Brachypodium hybridum, 35S rDNA IGS, FISH, 5S rDNA NTS, nrITS, rDNA loci,
• MeSH
• Brachypodium * genetika   MeSH
• fylogeneze * MeSH
• genetická variace MeSH
• genom rostlinný MeSH
• geny rRNA genetika   MeSH
• molekulární evoluce * MeSH
• polyploidie * MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ribozomální DNA MeSH
• RNA ribozomální 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
• fylogeneze MeSH
• genetická variace * MeSH
• houby genetika   MeSH
• molekulární evoluce MeSH
• mutace MeSH
• polymorfismus genetický * MeSH
• ribozomální DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ribozomální DNA 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.

• Klíčová slova
• Cycadaceae, centromeres, chromosome rearrangements, epigenetics, genome evolution, gymnosperms, telomeres,
• MeSH
• centromera genetika   MeSH
• cykasy * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• Magnoliopsida * genetika   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

• Klíčová slova
• allopolyploidy, genome size, genomic shock, marsh orchids, transposable elements,
• MeSH
• diploidie MeSH
• genom rostlinný MeSH
• lidé MeSH
• mokřady MeSH
• Orchidaceae * genetika   MeSH
• polyploidie MeSH
• sourozenci * MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transpozibilní elementy DNA MeSH

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.

• Klíčová slova
• 35S rDNA, 3D-FISH, Brachypodium, allopolyploidy, nucleolar dominance, rRNA gene expression, secondary constriction,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Brachypodium hybridum, 35S rDNA evolution, 35S rRNA gene expression, allopolyploidy, nucleolar dominance,
• MeSH
• Brachypodium genetika   metabolismus   MeSH
• chromozomy rostlin genetika   MeSH
• genetické lokusy genetika   MeSH
• genom rostlinný genetika   MeSH
• geny rRNA genetika   MeSH
• metylace DNA genetika   MeSH
• molekulární evoluce MeSH
• polymorfismus genetický genetika   MeSH
• rostlinné geny genetika   MeSH
• Southernův blotting MeSH
• tetraploidie * MeSH
• variabilita počtu kopií segmentů DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. 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.

• Klíčová slova
• bryophytes, cytosine methylation, epigenetics, genome evolution, histone marks, rDNA,
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND AIMS: Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. METHODS: We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. KEY RESULTS: Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH ('Darmor', 'Yudal' and 'Asparagus kale') harboured the same number (12 per diploid set) of loci. In B. napus 'Darmor', the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus 'Darmor'. In contrast, B. napus 'Yudal' showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus 'Asparagus kale' showed an intermediate pattern to 'Darmor' and 'Yudal'. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar ('Norin 9') showed co-dominance. CONCLUSIONS: The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion.

• Klíčová slova
• Brassica napus, allopolyploidy, chromosome evolution, gene conversion, rDNA,
• MeSH
• Brassica napus genetika   MeSH
• genetická variace genetika   MeSH
• genetické lokusy genetika   MeSH
• genová konverze genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• ribozomální DNA genetika   MeSH
• Southernův blotting MeSH
• stanovení celkové genové exprese MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ribozomální DNA MeSH

Reticulate evolution is characterized by occasional hybridization between two species, creating a network of closely related taxa below and at the species level. In the present research, we aimed to verify the hypothesis of the allopolyploid origin of hexaploid C. album s. str., identify its putative parents and estimate the frequency of allopolyploidization events. We sampled 122 individuals of the C. album aggregate, covering most of its distribution range in Eurasia. Our samples included putative progenitors of C. album s. str. of both ploidy levels, i.e. diploids (C. ficifolium, C. suecicum) and tetraploids (C. striatiforme, C. strictum). To fulfil these objectives, we analysed sequence variation in the nrDNA ITS region and the rpl32-trnL intergenic spacer of cpDNA and performed genomic in-situ hybridization (GISH). Our study confirms the allohexaploid origin of C. album s. str. Analysis of cpDNA revealed tetraploids as the maternal species. In most accessions of hexaploid C. album s. str., ITS sequences were completely or nearly completely homogenized towards the tetraploid maternal ribotype; a tetraploid species therefore served as one genome donor. GISH revealed a strong hybridization signal on the same eighteen chromosomes of C. album s. str. with both diploid species C. ficifolium and C. suecicum. The second genome donor was therefore a diploid species. Moreover, some individuals with completely unhomogenized ITS sequences were found. Thus, hexaploid individuals of C. album s. str. with ITS sequences homogenized to different degrees may represent hybrids of different ages. This proves the existence of at least two different allopolyploid lineages, indicating a polyphyletic origin of C. album s. str.

• MeSH
• Chenopodium album genetika   MeSH
• cytogenetické vyšetření metody   MeSH
• DNA rostlinná genetika   MeSH
• fylogeneze MeSH
• genetická variace genetika   MeSH
• genomika MeSH
• molekulární evoluce * MeSH
• polyploidie * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA rostlinná MeSH

Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5'-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.

• Klíčová slova
• bioinformatics, duplication, paralogy, poaceae, polyploidy,
• MeSH
• anotace sekvence MeSH
• fylogeneze * MeSH
• genom rostlinný MeSH
• genomika metody   MeSH
• haplotypy * MeSH
• hybridizace in situ fluorescenční MeSH
• jednonukleotidový polymorfismus MeSH
• lipnicovité klasifikace   genetika   MeSH
• otevřené čtecí rámce MeSH
• polyploidie * MeSH
• reprodukovatelnost výsledků MeSH
• ribozomální DNA MeSH
• RNA ribozomální genetika   MeSH
• rostlinné geny MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA, ribosomal, 45S MeSH Prohlížeč