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Background and Aims: Rye supernumerary (B) chromosomes have an accumulation mechanism involving the B subtelomeric domain highly enriched in D1100- and E3900-related sequences. In this work, the effects of heat stress during the early stages of male meiosis in 0B and +B plants were studied., Methods: In-depth cytological analyses of chromatin structure and behaviour were performed on staged rye meiocytes utilizing DAPI, fluorescence in situ hybridization and 5-methylcytosine immune labelling. Quantitative real-time PCR was used to measure heat effects on the expression of the Hsp101 gene as well as the 3·9- and 2·7-kb E3900 forms in various tissues and meiotic stages., Key Results and Conclusions: Quantitative real-time PCR established that heat induced equal up-regulation of the Hsp101 gene in 0B and 2B plants, with a marked peak in anthers with meiocytes staged at pachytene. Heat also resulted in significant up-regulation of E3900-related transcripts, especially at pachytene and for the truncated 2·7-kb form of E3900. Cytological heat-induced anomalies in prophase I, measured as the frequency of anomalous meiocytes, were significantly greater in 0B plants. Whereas telomeric sequences were widely distributed in a manner close to normal in the majority of 2B pachytene cells, most 0B meiocytes displayed abnormally clustered telomeres after chromosome pairing had occurred. Relevantly, bioinformatic analysis revealed a significantly high-density heat responsive cis regulatory sequence on E3900, clearly supporting stress-induced response of transcription for the truncated variant. Taken together, these results are the first indication that rye B chromosomes have implications on heat tolerance and may protect meiocytes against heat stress-induced damage., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The union of different genomes in the same nucleus frequently results in hybrid genotypes with improved genome plasticity related to both genome remodeling events and changes in gene expression. Most modern cereal crops are polyploid species. Triticale, synthesized by the cross between wheat and rye, constitutes an excellent model to study polyploidization functional implications. We intend to attain a deeper knowledge of dispersed repetitive sequence involvement in parental genome reshuffle in triticale and in wheat-rye addition lines that have the entire wheat genome plus each rye chromosome pair. Through Random Amplified Polymorphic DNA (RAPD) analysis with OPH20 10-mer primer we unraveled clear alterations corresponding to the loss of specific bands from both parental genomes. Moreover, the sequential nature of those events was revealed by the increased absence of rye-origin bands in wheat-rye addition lines in comparison with triticale. Remodeled band sequencing revealed that both repetitive and coding genome domains are affected in wheat-rye hybrid genotypes. Additionally, the amplification and sequencing of pSc20H internal segments showed that the disappearance of parental bands may result from restricted sequence alterations and unraveled the involvement of wheat/rye related repetitive sequences in genome adjustment needed for hybrid plant stabilization.

Polyploidization is one of the major driving forces in plant evolution and is extremely relevant to speciation and diversity creation. Polyploidization leads to a myriad of genetic and epigenetic alterations that ultimately generate plants and species with increased genome plasticity. Polyploids are the result of the fusion of two or more genomes into the same nucleus and can be classified as allopolyploids (different genomes) or autopolyploids (same genome). Triticeae synthetic allopolyploid species are excellent models to study polyploids evolution, particularly the wheat-rye hybrid triticale, which includes various ploidy levels and genome combinations. In this review, we reanalyze data concerning genomic analysis of octoploid and hexaploid triticale and different synthetic wheat hybrids, in comparison with other polyploid species. This analysis reveals high levels of genomic restructuring events in triticale and wheat hybrids, namely major parental band disappearance and the appearance of novel bands. Furthermore, the data shows that restructuring depends on parental genomes, ploidy level, and sequence type (repetitive, low copy, and (or) coding); is markedly different after wide hybridization or genome doubling; and affects preferentially the larger parental genome. The shared role of genetic and epigenetic modifications in parental genome size homogenization, diploidization establishment, and stabilization of polyploid species is discussed.

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat-rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat-rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale x wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat-rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat-rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.

Two sequence families, E3900 and D1100, are amplified on the subtelomeric domain of the long arm of rye B chromosomes, the region that controls its drive mechanism. In this work, polymerase chain reaction (PCR) with a number of primers spanning E3900 shows that the organization and nucleotide sequence of E3900-related portions are present and highly conserved on rye A chromosomes as well as in other cereals. Quantitative Real-Time PCR estimates two E3900 sequences to be represented in 100-150 copies on Bs and at least as single copies on As. A novel E3900-related sequence, with a deletion that results in a frameshift and subsequently an open reading frame with putative DNA binding motifs, is identified. Expression analysis of E3900 indicates identical transcription levels in leaves from plants with and without Bs, showing that the expression of these sequences must be silenced on Bs and tightly regulated on As in leaves. In contrast, E3900 transcription is upregulated during meiosis exclusively in plants with Bs, maintaining a high level of transcription in the gametophyte. Interestingly, Bs not only influence their own chromatid segregation but also that of the regular chromosome complement of both rye and wheat. There is a drastic increase in frequency of disrupted metaphase and anaphase cells in the first mitosis of pollen grains carrying Bs, which appears to be due to anomalous adherences between sister chromatids. Taken together, this work provides insight into how E3900 sequences are potentially associated with important evolutionary mechanisms involved in basic cellular processes.

Background: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions., Methodology/principal Findings: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs., Conclusions/significance: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to 'mutual amphiplasty' in the nucleolar dominance process.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.

The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

'Lindström' wheat (AABBDD+rye B chromosomes) was used to study the effects of alien chromatin introgressed into a wheat genetic background, subjecting the wheat genome to a new and transient allopolyploidisation episode. Using this experimental material, we have previously demonstrated that no large-scale chromosomal translocations occurred as a result of the genomic constitution of the addition line. However, we have shown that the presence of a number of rye B chromosomes is associated with changes in the interphase organization and expression patterns of wheat rDNA loci. We have now extended our studies to focus on a further characterization of 'Lindström' 5S rDNA loci and also on high molecular weight glutenin subunit (HMW-GS) patterns. In the process, we have uncovered an unusually large variant of the 5S rDNA locus on wheat chromosome 1B (not to be confused with rye B chromosomes) and 2 novel HMW glutenin y-type alleles. These changes are not directly related to variation in rye B chromosome number in the present material, but the fact that a new, and still segregating, 1Dy HMW-GS gene was identified indicates a recent timescale for its origin. Strikingly, the 'Lindström' 5S rDNA 1B locus integrates a unit sharing 94% homology with a rye 5S rDNA sequence, suggesting the possibility that the wheat locus was colonized by highly homologous rye sequences during the breeding of 'Lindström', when the rye and wheat genomes were together, albeit briefly, in the same nucleus.

Spontaneous DNA insertions from supernumerary B chromosomes (Bs) into the standard A chromosome complement were detected in rye (Secale cereale L.), using fluorescent in situ hybridization (FISH) analysis with the D1100 B-specific sequence probe. The insertions were seen in individuals derived from plants possessing deleted Bs, characterized in this study by not having the B-specific sequences that are normally found at the distal part of the long arm of the standard rye B. This result supports the case for the spontaneous introgression of B-specific DNA into the A chromosome genome, and it indicates that 'cross-talking' between A and B chromosomes may occur in wild populations.

The standard rye cultivar 'Imperial' and a structural variant carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms) were used to investigate expression patterns of homologous rDNA loci, and the influence of chromosome structural change on their interphase organisation and relative disposition. Sequential silver staining and in situ hybridization with the rDNA probe pTa71, established a correspondence between the expression and organization patterns of rDNA domains in metaphase and interphase cells. In most cells of the cultivar Imperial, nucleolar organizer region (NOR) silver staining on metaphase chromosomes with equivalent numbers of rDNA genes revealed a size heteromorphism between homologous rDNA loci, resulting from their differential expression. NOR heteromorphism in the structural variant line was significantly reduced. The preferential activity of one NOR over its homologue was found to be random within cells and independent of parental origin. Nucleotypic modifications mediated by changes in the 1R chromosome structure include increased proximity between homologous rDNA loci in interphase, and an increase in the frequency of cells with intra-nucleolar ribosomal condensed chromatin. These results seem to indicate a 'sequence recognition' process for the regulation of homologous loci.