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

Genome or genomic dominance (GD) is a phenomenon observed in hybrids when one parental genome becomes dominant over the other. It is manifested by the replacement of chromatin of the submissive genome by that of the dominant genome and by biased gene expression. Nucleolar dominance (ND) - the functional expression of only one parental set of ribosomal genes in hybrids - is another example of an intragenomic competitive process which, however, concerns ribosomal DNA only. Although GD and ND are relatively well understood, the nature and extent of their potential interdependence is mostly unknown. Here, we ask whether hybrids showing GD also exhibit ND and, if so, whether the dominant genome is the same. To test this, we used hybrids between Festuca and Lolium grasses (Festulolium), and between two Festuca species in which GD has been observed (with Lolium as the dominant genome in Festulolium and F. pratensis in interspecific Festuca hybrids). Using amplicon sequencing of ITS1 and ITS2 of the 45S ribosomal DNA (rDNA) cluster and molecular cytogenetics, we studied the organization and expression of rDNA in leaf tissue in five hybrid combinations, four generations and 31 genotypes [F. pratensis × L. multiflorum (F1, F2, F3, BC1), L. multiflorum × F. pratensis (F1), L. multiflorum × F. glaucescens (F2), L. perenne × F. pratensis (F1), F. glaucescens × F. pratensis (F1)]. We have found that instant ND occurs in Festulolium, where expression of Lolium-type rDNA reached nearly 100% in all F1 hybrids and was maintained through subsequent generations. Therefore, ND and GD in Festulolium are manifested by the same dominant genome (Lolium). We also confirmed the concordance between GD and ND in an interspecific cross between two Festuca species.

• Klíčová slova
• Festuca, Lolium, fluorescent in situ hybridization, genome dominance, genomic in situ hybridization, internal transcribed spacer, nucleolar dominance, ribosomal DNA,
• Publikační typ
• časopisecké články 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