In situ hybridization of ribosomal DNA to rose chromosomes

The genus Rosa consists of approximately 200 species and 20,000 cultivars, most of complex hybrid origin. Few of the current cultivars, most of which are tetraploid with 2n = 4x = 28, can be traced unambiguously to their wild diploid progenitor species. Fluorescent in situ hybridization (FISH) offers the possibility to estimate the proportion of various donor genomes in complex advanced-generation hybrids. To establish the feasibility of FISH in rose and to explore differences, if any, in number and subchromosomal location of nucleolar organizer regions (NORs), mitotic chromosome spreads from five diploid species and a commercial tetraploid cultivar were probed with part of the 18S-26S rDNA repeat from soybean. Shoot tips were the most convenient source of mitotic divisions for FISH. Cell suspensions from enzymatically digested shoot tips were spread with 3 ethanol:1 acetic acid on glass slides and then squashed in 45% acetic acid under a coverslip. Prolonged digestion in cellulase and pectolyase was required to obtain satisfactory chromosome spreading and exposure for FISH. A pair of strong, subterminal hybridization signals, corresponding to a single NOR per genome, was observed in the diploid species. The commercial tetraploid rose cultivar had four strong, subterminal signals and thus one NOR per genome. However, two of the signals were consistently stronger than the other two. The results demonstrate the feasibility of detecting repetitive sequences in spread chromosomes of roses.