Switch them off or not: selective rRNA gene repression in grasses.

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.

Cytomolecular Organisation of the Nuclear Genome.

Modern molecular cytogenetics allows many aspects of the nuclear genome structure, function, and evolution to be analysed within the topographic context of mitotic and meiotic chromosomes and interphase nuclei [...].

Corrigendum: To Be or Not to Be Expressed: The First Evidence of a Nucleolar Dominance Tissue-Specificity in Brachypodium hybridum.

[This corrects the article DOI: 10.3389/fpls.2021.768347.].

To Be or Not to Be Expressed: The First Evidence of a Nucleolar Dominance Tissue-Specificity in Brachypodium hybridum.

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.

Novel visual analytics approach for chromosome territory analysis.

This document presents a new and improved, more intuitive version of a novel method for visually representing the location of objects relative to each other in 3D. The motivation and inspiration for developing this new method came from the necessity for objective chromosome territory (CT) adjacency analysis. The earlier version, Distance Profile Chart (DPC), used octants for 3D orientation. This approach did not provide the best 3D space coverage since space was divided into just eight cones and was not intuitive with regard to orientation in 3D. However, the version presented in this article, called DPC12, allows users to achieve better space coverage during conification since space is now divided into twelve cones. DPC12 is faster than DPC and allows for a more precise determination of the location of objects in 3D. In this article a short introduction about the conification idea is presented. Then we explain how DPC12 is designed and created. After that, we show DPC12 on an instructional dataset to make it easier to understand and demonstrate how they appear and how to read them. Finally, using DPC12 we present an example of an adjacency analysis (AA) using the model of Chromosome Territories (CTs) distribution in the rice nucleus.

The fate of 35S rRNA genes in the allotetraploid grass Brachypodium hybridum.

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.

Ribosomal DNA loci derived from Brachypodium stacei are switched off for major parts of the life cycle of Brachypodium hybridum.

Nucleolar dominance is an epigenetic phenomenon that occurs in some plant and animal allopolyploids and hybrids, whereby only one ancestral set of 35S rRNA genes retains the ability to form the nucleolus while the rDNA loci derived from the other progenitor are transcriptionally silenced. There is substantial evidence that nucleolar dominance is regulated developmentally. This study focuses upon the establishment and/or maintenance of nucleolar dominance during different stages of development in the model grass allotetraploid Brachypodium hybridum. Fluorescence in situ hybridization with a 25S rDNA probe to cells in three-dimensional cytogenetic preparations showed that nucleolar dominance is present not only in root meristematic and differentiated cells of this species, but also in male meiocytes at prophase I, tetrads of microspores, and different embryonic tissues. The inactive state of Brachypodium stacei-originated rDNA loci was confirmed by silver staining. Only B. distachyon-derived 35S rDNA loci formed nucleoli in the aforementioned tissues, whereas B. stacei-like loci remained highly condensed and thus transcriptionally suppressed. The establishment of nucleolar dominance during earlier stages of B. hybridum embryo development cannot be ruled out. However, we propose that gradual pseudogenization of B. stacei-like loci in the evolution of the allotetraploid seems to be more likely.

The arrangement of Brachypodium distachyon chromosomes in interphase nuclei.

The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biological processes, such as DNA replication, transcription, and repair. However, detailed data about the CT arrangement in monocotyledonous plants are scarce. In this study, chromosome painting was used to analyse the distribution and associations of individual chromosomes in the 3-D preserved nuclei of Brachypodium distachyon root cells in order to determine the factors that may have an impact on the homologous CT arrangement. It was shown that the frequency of CT association is linked to the steric constraints imposed by the limited space within the nucleus and may depend on chromosome size and morphology as well as on the nuclear shape. Furthermore, in order to assess whether the distribution of interphase chromosomes is random or is subject to certain patterns, a comparison between the experimental data and the results of a computer simulation (ChroTeMo), which was based on a fully probabilistic distribution of the CTs, was performed. This comparison revealed that homologous chromosome arm CTs associate more often than if they were randomly arranged inside the interphase nucleus.

Chromosome Territory Modeller and Viewer.

This paper presents ChroTeMo, a tool for chromosome territory modelling, accompanied by ChroTeVi-a chromosome territory visualisation software that uses the data obtained by ChroTeMo. These tools have been developed in order to complement the molecular cytogenetic research of interphase nucleus structure in a model grass Brachypodium distachyon. Although the modelling tool has been initially created for one particular species, it has universal application. The proposed version of ChroTeMo allows for generating a model of chromosome territory distribution in any given plant or animal species after setting the initial, species-specific parameters. ChroTeMo has been developed as a fully probabilistic modeller. Due to this feature, the comparison between the experimental data on the structure of a nucleus and the results obtained from ChroTeMo can indicate whether the distribution of chromosomes inside a nucleus is also fully probabilistic or is subjected to certain non-random patterns. The presented tools have been written in Python, so they are multiplatform, portable and easy to read. Moreover, if necessary they can be further developed by users writing their portions of code. The source code, documentation, and wiki, as well as the issue tracker and the list of related articles that use ChroTeMo and ChroTeVi, are accessible in a public repository at Github under GPL 3.0 license.

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species.

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere-telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed.