-Artificial chromosome duplication of diploid genotypes of

Turfgrass cultivation is a multi-purpose popular practice, associated with life quality of urban populations in consequence of its capability of providing safe, pleasant environments, suitable for recreational and sportive activities. Additionally, it plays relevant environmental functions. For these reasons, turfgrasses constitute an important segment of the agribusiness in many countries. In Brazil, the importance of this type of cultivation has increased in spite of the reduced number of available turfgrass cultivars. Research programs aiming the exploitation of his ample genetic diversity could promote Brazil to the forefront of this type of agriculture in the tropical world. In this work, we discussed and proposed degrees of relevance for different characteristics of grass plants destined for use as permanent ground coverage under a variety of Brazilian situations.

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.

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.

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.