Microimage software

Giemsa-stained chromosomes and FISH images were inspected using a Provis AX70 Olympus microscope with a standard fluorescence filter set. FISH images were captured under immersion objective 100× with a black and white CCD camera (DP30W Olympus) for each fluorescent dye using Olympus Acquisition Software. The digital images were then pseudocoloured (blue for DAPI, red for Rhodamine or Cy3, green for FITC or Alexa488) and superimposed with MicroImage software (Olympus, version 4.0). FISH karyotype images were optimized and arranged using Adobe Photoshop, version CS6. Karyotypes from Giemsa-stained and C-banded images were arranged in IKAROS (Metasystems) software.

Mitotic metaphase spreads were prepared according to Doleželová et al. [17 ]. Actively growing root tips were pre-treated in 0.05% (w/v) 8-hydroxyquinoline for 3 hrs at room temperature and then fixed in 3:1 ethanol: acetic acid overnight. Fixed roots were washed in a solution of 75 mM KCl and 7.5 mM EDTA (pH 4) and meristem tips were digested in a mixture of 2% (w/v) pectinase and 2% (w/v) cellulase in 75 mM KCl and 7.5 mM EDTA (pH 4) for 90 min at 30°C. Protoplast suspension was then filtered through a 150 μm nylon mesh and pelleted. The pellet was resuspended in 75 mM KCl and 7.5 mM EDTA (pH 4) and incubated for 5 min at room temperature. After pelleting, the protoplasts were washed three times with 70% ethanol. Five μl of the suspension were dropped onto a slide and shortly before drying out, 5 μl of 3:1 fixative were added to the drop to induce protoplast bursting. Finally, the slide was rinsed in 100% ethanol and air-dried.
For chromosome counting, the preparations were stained with DAPI (Vectashield Mounting Medium with DAPI; Vector laboratories). Slides were examined with Olympus AX70 fluorescence microscope. Images were captured using a cooled high-resolution black and white camera and processed using MicroImage software (Olympus, Tokyo, Japan). In each plant, two slides were observed, each with at least five metaphase plates.

Probes for 45S rDNA and 5S rDNA were prepared by labeling Radka1 DNA clone (45S rDNA) and Radka2 DNA clone (5S rDNA) [44 (link)] with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science). Both probes were labeled by PCR using M13 forward and reverse primers (Invitrogen). Hybridization mixture consisting of 50% formamide, 10% dextran sulfate in 1×SSC and 1 μg/ml of each labeled probe was added onto slides and denatured at 80°C for 3 min. The hybridization was carried out at 37°C overnight. The sites of probe hybridization were detected using anti-digoxigenin-FITC (Roche Applied Science) and streptavidin-Cy3 (Vector Laboratories, Burlingame, USA), and the chromosomes were counterstained with DAPI. The slides were examined with Olympus AX70 fluorescence microscope and the images of DAPI, FITC and Cy-3 fluorescence were acquired separately with a cooled high-resolution black and white CCD camera. The camera was interfaced to a PC running the MicroImage software (Olympus, Tokyo, Japan). At least ten complete metaphases were examined for every accession.