Repetitive DNA contributes significantly to plant genome size, adaptation, and evolution. However, little is understood about the transcription of repeats. This is addressed here in the plant green foxtail millet (Setaria viridis). First, we used RepeatExplorer2 to calculate the genome proportion (GP) of all repeat types and compared the GP of long terminal repeat (LTR) retroelements against annotated complete and incomplete LTR retroelements (Ty1/copia and Ty3/gypsy) identified by DANTE in a whole genome assembly. We show that DANTE-identified LTR retroelements can comprise ∼0.75% of the inflorescence poly-A transcriptome and ∼0.24% of the stem ribo-depleted transcriptome. In the RNA libraries from inflorescence tissue, both LTR retroelements and DNA transposons identified by RepeatExplorer2 were highly abundant, where they may be taking advantage of the reduced epigenetic silencing in the germ line to amplify. Typically, there was a higher representation of DANTE-identified LTR retroelements in the transcriptome than RepeatExplorer2-identified LTR retroelements, potentially reflecting the transcription of elements that have insufficient genomic copy numbers to be detected by RepeatExplorer2. In contrast, for ribo-depleted libraries of stem tissues, the reverse was observed, with a higher transcriptome representation of RepeatExplorer2-identified LTR retroelements. For RepeatExplorer2-identified repeats, we show that the GP of most Ty1/copia and Ty3/gypsy families were positively correlated with their transcript proportion. In addition, guanine- and cytosine-rich repeats with high sequence similarity were also the most abundant in the transcriptome, and these likely represent young elements that are most capable of amplification due to their ability to evade epigenetic silencing.

• MeSH
• Transcription, Genetic MeSH
• Genome, Plant MeSH
• Terminal Repeat Sequences * MeSH
• Gene Expression Regulation, Plant MeSH
• Retroelements * MeSH
• Setaria Plant * genetics   MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Retroelements * 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

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

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

• MeSH
• Cytogenetic Analysis methods   MeSH
• Genome MeSH
• In Situ Hybridization, Fluorescence MeSH
• Chromosome Mapping MeSH
• Evolution, Molecular * MeSH
• Butterflies genetics   MeSH
• Moths genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Small Nuclear genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• RNA, Ribosomal, 5S 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
• RNA, Small Nuclear MeSH
• RNA, Ribosomal, 18S MeSH
• RNA, Ribosomal, 5S MeSH
• U2 small nuclear RNA MeSH Browser

Nuclear ribosomal DNA (nrDNA) has displayed extraordinary dynamics during the evolution of plant species. However, the patterns and evolutionary significance of nrDNA array expansion or contraction are still relatively unknown. Moreover, only little is known of the fate of minority nrDNA copies acquired between species via horizontal transfer. The barley genus Hordeum (Poaceae) represents a good model for such a study, as species of section Stenostachys acquired nrDNA via horizontal transfer from at least five different panicoid genera, causing long-term co-existence of native (Hordeum-like) and non-native (panicoid) nrDNAs. Using quantitative PCR, we investigated copy number variation (CNV) of nrDNA in the diploid representatives of the genus Hordeum. We estimated the copy number of the foreign, as well as of the native ITS types (ribotypes), and followed the pattern of their CNV in relation to the genus' phylogeny, species' genomes size and the number of nrDNA loci. For the native ribotype, we encountered an almost 19-fold variation in the mean copy number among the taxa analysed, ranging from 1689 copies (per 2C content) in H. patagonicum subsp. mustersii to 31342 copies in H. murinum subsp. glaucum. The copy numbers did not correlate with any of the genus' phylogeny, the species' genome size or the number of nrDNA loci. The CNV was high within the recognised groups (up to 13.2 × in the American I-genome species) as well as between accessions of the same species (up to 4×). Foreign ribotypes represent only a small fraction of the total number of nrDNA copies. Their copy numbers ranged from single units to tens and rarely hundreds of copies. They amounted, on average, to between 0.1% (Setaria ribotype) and 1.9% (Euclasta ribotype) of total nrDNA. None of the foreign ribotypes showed significant differences with respect to phylogenetic groups recognised within the sect. Stenostachys. Overall, no correlation was found between copy numbers of native and foreign nrDNAs suggesting the sequestration and independent evolution of native and non-native nrDNA arrays. Therefore, foreign nrDNA in Hordeum likely poses a dead-end by-product of horizontal gene transfer events.

• Keywords
• copy number variation (CNV), fluorescent in situ hybridisation (FISH), horizontal gene transfer (HGT), internal transcribed spacer (ITS), nuclear ribosomal DNA (nrDNA), phylogeny, qPCR (quantitative PCR), xenolog,
• Publication type
• Journal Article 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