Characterization Of The 45S RDNA Fragile Site In Ryegrass

Ribosomal DNAs (rDNAs) are highly conserved and play a very important role in organisms, which are organized in tandem arrays in some pairs of chromosomes. The karyotype of Lolium spp. was analyzed using metaphase chromosomes by fluorochrome DAPI banding and FISH with 45S and 5S rDNA probes. From the karyotype of three ryegrass cultivars (dioploid'Accent', tetraploid'Top One'and'Bison'), it was found that the dark region of DAPI straining was coincident with the centromere region and rDNA flanking region. Each pair of homologous chromosomes had a unique fluorescence banding pattern, which was analogous in number, size and distribution of bands. The DAPI banding patterns within a species were stable and could be used for identifying the homologous chromosomes. The result also showed that the number of 45S rDNA varied remarkably. Seven 45S rDNA loci were detected on chromosomes of diploid ryegrass'Accent', while 10 and 11 loci were found in tetraploid ryegrass'Top One'and'Bison' respectively. We speculated that some 45S rDNA locus were lost in the evolution of polyploidization. However, the chromosomal distribution patterns of 45S rDNA were highly conserved, usually located on the long arm and near centromere. However the distribution of 5S rDNA loci on chromosomes was highly variable and the number of 5S rDNA loci was conserved. In tetraploid, one pair of hybridization signals was detected on the long arm and another pair of signals on the short arm while 5S rDNA in diploid ryegrass'Accent'occurred on the short arm chromosome 3. The establishment of ryegrass rDNA-DAPI banding kayrotype would lay a foundation for the further cytogenetic research such as chromosomal structure analysis and physical mapping of genes,breeding and the resource use in Lolium spp.During the course of establishing the ryegrass rDNA-DAPI banding kayrotype, we found that the number of chromosomes (actually plus chromosome fragments) was often more than the expected 14 in most cells for dioploid and 28 for tetraploid. The further close cytological examination using a routine chromosome preparation procedure reveald that 85% of spreads contained more than 14 chromosomes in'Player'. Fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome breakage) at 45S rDNA sites, and all breakage occurred at the sites of 45S rDNA in'Player', as well as in'Top One'. Three different cytological appearances of breakage were observed at the 45S rDNA sites in Lolium:breakage occurred to a single chromatid; gap within the rDNA with the chromosome still connected through one or a few thin DNA fibers; no detectable DNA hybridization signals between the broken ends of the two chromosome fragments. The chromosome breakage observed in this study is cytologically very similar to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency (86%) of chromosome breakage in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Chromatin breakage occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. Spontaneous fragile site expression in the form of chromosome breakage in high-frequency without any treatment suggesting the fragility is the unique structural characteristics of ryegrass fragile sites. The characterization of the 45S rDNA fragile site in ryegrass would extend the understanding of the 45S rDNA and chromosome fragile sites, and possibly provide a new model for the studies of fragile sites in human.FISH in flow-sorted G2/M interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was further studied by atomic force microscope (AFM) combining with FISH analysis. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatin which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55-65 nm, composed of just two 30-nm fibers of chromatin. AFM topography images of another two gaps showed that the thickness along connected DNA fibers was not uniform, which indicated that the different degrees of chromatin folding occurred at different locations within the 45S rDNA regions. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in chromosome breakage under the optical microscope.