nucleolus organizer region activation by 5-azacytidine in wheat x rye hybrids. Genome, 33: 707-712. Nucleolar activity was studied in several lines of Triticum aestivum cv. Chinese Spring, Triticum turgidum cv. Durum, and F1 hybrids from euploid and aneuploid lines of T. aestivum and Secale cereale cv. Centeio d o Alto, in cells from root tips of seeds germinated in water or in 5-azacytidine. 5-Azacytidine, an analog of cytidine modified in the 5 position of the pyrimidine ring, inhibits DNA methylation. By using silver staining to determine the number of nucleolus organizer regions and the average number of nucleoli per root-tip cell from seeds germinated in both situations, it became apparent that the presence of 5-azacytidine during germination allowed for the expression of the nucleolus organizer region locus belonging to the rye genome, in contrast to the usual observed cytological absence of the rye nucleolus organizer region in wheat-rye hybrids. It is suggested that wheat nucleolar dominance in wheat-rye hybrids is mainly a consequence of methylation of rRNA genes or its regulators located on the 1R chromosome of rye.

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.

The expression of rRNA genes located in the nucleolar organizing region (NOR) present on the short arm of chromosome 1R from rye (Secale cereale L.) was examined in several hexaploid (Triticum aestivum L.) and tetraploid wheats (Triticum turgidum L.) containing the entire chromosome 1R from rye (disomic substitution 1B(1R)), its full haploid genome (hexaploid wheat–rye F1 hybrid), or only its short arm translocated to the long arms of wheat chromosomes from the homoeologous group 1 (disomic translocations 1AL/1RS, 1BL/1RS, or 1DL/1RS) or added to the complete hexaploid wheat genotype (monotelosomic addition 1RS). By silver staining and determination of the number of Ag-NORs and the average number of nucleoli per root-tip cell it became apparent that the expression of 1R NORs, in the presence of wheat genomes, depends on the absence of the long arm of rye chromosome 1R. In wheat-rye F1 hybrids and in hexaploid wheat with a disomic substitution 1B(1R), 1R NOR was morphologically absent, even when only one wheat major NOR was present, in contrast with its frequent expression in wheat–rye translocation or addition lines where only its short arm was added. It is suggested that wheat nucleolar dominance over rye as expressed by heterochromatic and silent NOR in 1RS is under a complex genetic control which involves interaction between 1RL and unidentified wheat genes.Key words: 1R nucleolus organizer region, gene activity, amphiplasty.

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The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret 1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret 1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret 1 distribution between cultivars. The combined cytological and molecular results suggest that Gret 1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.

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.

To test the hypothesis that interspecific genomic and chromosome interactions leading to nucleolar dominance could be reprogrammed in meiosis, we compared the expression of distinct nucleolar organizing region (NOR) loci in hexaploid triticale root tip meristematic cells, pollen mother cells and young pollen grains. Interphase and metaphase cells were silver stained to quantify nucleoli and active NOR loci respectively. A marked difference in the ribosomal RNA gene activity of each locus was observed when different types of cells were compared: in somatic and pollen mother cells, rRNA gene activity was mainly restricted to major wheat NORs (1B and 6B) with only a small contribution from rye NORs (1R). In contrast, in young pollen grains, all NORs present, including the 1R NORs, were consistently active. The expression of all NORs just after meiosis is considered to be a consequence of meiotic reprogramming of rye origin rDNA. Gene reprogramming mediated by the resetting of methylation patterns established early in embryogenesis is suggested to be responsible for the differential expression of the NORs of rye origin in distinct developmental stages of triticale.

The economic and ecological importance of forest trees, as well as their unique biological features, has recently raised the level of interest in studies on their genomes, including sequencing of the entire poplar genome. However, cytogenetic studies have not moved in parallel with developments in genomics. This is especially true for hardwood species characterized by small genomes and relatively high numbers of small chromosomes. Molecular cytogenetic studies have mainly been focused on coniferous species, owing to the larger size of their chromosomes, and have been applied exclusively for chromosome identification and comparative karyotyping in an attempt to understand genome evolution and phylogenetic relationships. In this context, rRNA genes physical mapped by FISH reveal particularly useful chromosomal landmarks with variable distribution patterns between species. Here we present a contribution of DNA markers used for chromosome analysis, which already allowed a deeper characterization and understanding of the processes underlying genome diversity of forest trees. The use of advanced cytogenetic techniques and other potential important methods for genome analysis of forest trees is also discussed.