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Rocha L.C.,Federal University of Lavras | Mittelmann A.,Embrapa Dairy Cattle Embrapa Temperate Agriculture | Houben A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Techio V.H.,Federal University of Lavras
Molecular Biology Reports

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages. © 2016, Springer Science+Business Media Dordrecht. Source

Bustamante F.O.,Federal University of Lavras | Rocha L.C.,Federal University of Lavras | Torres G.A.,Federal University of Lavras | Davide L.C.,Federal University of Lavras | And 2 more authors.
Crop Science

The distribution, number, and location of ribosome DNA (rDNA) regions on chromosomes were evaluated, and transcriptional activity was described in genotypes of Italian ryegrass (Lolium multiflorum Lam.), as well as in an offspring resulting from interbreeding. Nucleoli and nucleolar organizer regions were labeled with silver nitrate and fluorescent in situ hybridization (FISH) was performed with 5S and 45S rDNA as probes. Hybridization procedures were performed on slides previously stained with the Ag-NOR method. The 5S rDNA sites were highly conserved, while 45S rDNA sites had wide variability, even showing more than one site on the same chromosome. One of the genotypes had more than expected 45S rDNA sites. Approximately 95% of the cells at metaphases had at least one chromosome break/gap in the 45S rDNA site, resulting in chromosome fragmentation. Thus, 45S rDNA correspond to fragile sites in L. multiflorum. These events can affect genome organization and cause new chromosomal rearrangements which, along with some other factors, might be responsible for microevolutionary changes involved in differentiation and speciation. Not all 45S rDNA sites are transcriptionally active. Variation in both the number and size of nucleoli and mechanisms of nucleolar fusion were observed in L. multiflorum. © Crop Science Society of America. Source

Rocha L.C.,Minas Gerais State University | Jankowska M.,Leibniz Institute of Plant Genetics and Crop Plant Research | Fuchs J.,Leibniz Institute of Plant Genetics and Crop Plant Research | Mittelmann A.,Embrapa Dairy Cattle Embrapa Temperate Agriculture | And 2 more authors.

Fragile sites (FSs) in plants have been described for species like Lolium and other grasses. Whereas in humans FSs were shown to be involved in genome instabilities; the consequences of FSs expression in plants are not known yet. To evaluate whether FSs cause karyotype instabilities, we assessed the frequency of micronuclei and lagging chromosomes in meristematic cells, the stability of the DNA content, and the occurrence of neocentromeres in the presumed chromosomal fragments of Lolium perenne, Lolium multiflorum, Festuca arrundinacea, and two Festulolium hybrids. The cell cycle analysis along with flow cytometric genome size measurements showed high stability in all genomes evaluated. Neocentromeric activity was neither observed in the presumed fragments nor in any other chromosomal region, then this is not the mechanism responsible by the stability. However, Fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe in combination with YOYO staining of metaphasic chromosomes showed that many extended nucleolus organizing region (NOR) form very thin YOYO-positive chromatin fibers connecting the acentric ‘fragment’ with the centromere-containing chromosome region. The obtained data indicate that the expression of FSs does not result in genome instabilities or neocentromere formation. The FS-containing 45S rDNA carrying chromatin fibers undergo a cell cycle and gene activity-dependent dynamic decondensation process. © 2016 Springer-Verlag Wien Source

Rocha L.C.,Federal University of Lavras | de Oliveira Bustamante F.,Federal University of Lavras | Silveira R.A.D.,Federal University of Lavras | Torres G.A.,Federal University of Lavras | And 2 more authors.

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA+ bands were colocalized with the 45S sites. © 2014 Springer-Verlag Wien. Source

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