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

Abstract

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.

Fig. 1.

Localization of B. distachyon- and B. stacei-inherited 35S rDNA loci (red fluorescence) in 3D cytogenetic preparations of B. hybridum prophase I meiocytes. Selected sections that contain 25S rDNA hybridization signals are shown. (A) Zygotene. (B) Pachytene. (C, D) Two different sections of one nucleus at diplotene. (E) Diakinesis. (F) Diagram showing the localization and condensation state of 35S rDNA loci in B. hybridum at different stages of mitosis and meiosis. Bd, B. distachyon-like 35S rDNA loci; Bs, B. stacei-like 35S rDNA loci; Nu, nucleolus. Sections are counterstained with DAPI (blue fluorescence). Scale bars=5 µm.

Fig. 2.

Transcriptional activity and DNA methylation pattern of 35S rDNA loci in B. hybridum meiocytes at pachytene. (A, B) Sequential FISH with 25S rDNA (red fluorescence) as a probe (A) and silver staining (B) on a representative cell at pachytene. Black arrows indicate Ag-NORs. (C–E) Sequential FISH with 25S rDNA (red fluorescence) as a probe (C) and immunolocalization of 5-MeC (D) on a representative pachytene. (E) Superimposed images C and D. Yellow arrows indicate the position of B. stacei-inherited 35S rDNA loci. Bd, B. distachyon-like 35S rDNA loci; Bs, B. stacei-like 35S rDNA loci; Nu, nucleolus. Bivalents are counterstained with DAPI (grey). Scale bars=5 µm.

Fig. 3.

Localization of B. distachyon- and B. stacei-inherited 35S rDNA loci (red fluorescence) in 3D cytogenetic preparations of B. hybridum microspores. Selected sections containing 25S rDNA hybridization signals are shown. Yellow arrows indicate the position of B. stacei-inherited 35S rDNA loci. Sections are counterstained with DAPI (grey). Scale bars=5 µm.

Fig. 4.

Distribution of 35S rDNA loci in nuclei/prometaphase chromosomes from different tissues of the B. hybridum embryo at BBCH83. (A) Longitudinal cross-section through a whole embryo. (B) Enlargement of the radicle and a fragment of coleorhiza. (C) Enlargement of shoot primordium and coleoptile. (D–K) FISH with 25S rDNA (red fluorescence) as a probe in nuclei from different embryo tissues. Selected sections that contain 25S rDNA hybridization signals and one or more nucleoli are presented. Bd, B. distachyon-like 35S rDNA loci; Bs, B. stacei-like 35S rDNA loci; cl, coleoptile; cr, coleorhiza; e, epiblast; ec, epithelial cells; Nu, nucleolus; r, radicle; sc, scutellum; Spr, shoot primordium. Sections are counterstained with DAPI (blue fluorescence). Scale bars (A–C)=50µm; (D–K)=5 µm.

Fig. 5.

Distribution of 35S rDNA loci in nuclei from different tissues of the B. hybridum embryo at BBCH01. (A) Longitudinal cross-section through a whole embryo. (B) Enlargement of the radicle and root cap. (C) Enlargement of the shoot primordium, leaf primordia, and coleoptile. (D–M) FISH with 25S rDNA (red fluorescence) as a probe in nuclei from different embryo tissues. Selected sections that contain 25S rDNA hybridization signals and one or more nucleoli are presented. Bd, B. distachyon-like 35S rDNA loci; Bs, B. stacei-like 35S rDNA loci; cl, coleoptile; cr, coleorhiza; e, epiblast; ec, epithelial cells; lp, leaf primordia; Nu, nucleolus; r, radicle; rc, root cap; sc, scutellum; Spr, shoot primordium. Sections are counterstained with DAPI (blue fluorescence). Scale bars (A–C)=50µm; (D–M)=5 µm.