Anti digoxigenin rhodamine conjugate

We randomly selected five good metaphases per individual and five individuals per population for constructing an idiogram. We selected three good metaphases from different individual per population for FISH. Root tips were obtained from seeds germinating in Petri dishes. The root tips were pretreated with 0.05% colchicine for 3 h at room temperature. The meristems were fixed in 3:1 ethanol: acetic acid for 24 h at room temperature and stored at -20°C until used for FISH. The FISH procedure was based on Zhang & Sang [17 (link)] with minor modifications. Probes used for FISH were 18S rDNA and 5S rDNA from PCR amplification, labeled using a DIG DNA Labeling and Detection Kit (Boehringer, Mannheim, Germany) with biotin-11-dUTP (Sigma) and digoxigenin-11-dUTP (Roche Diagnostics GmbH, Mannheim, Germany), respectively. The biotinylated-probes were detected by avidin-FIFC (Roche Diagnostics GmbH, Mannheim, Germany) and the digoxigenin-labelled probes by anti-digoxigenin rhodamine conjugate (Roche Diagnostics GmbH, Mannheim, Germany). All preparations were counterstained with DAPI (20 μg mL^-1), mounted in FluoroGuard^™ antifade reagent and observed with a Leica DMRBE microscope (Leica, Wetzlar, Germany). Fluorescent signals were captured by a SPOT cooled color digital camera system (Diagnostic instruments Inc., MI, USA).

Fluorescence in situ hybridization (FISH) was performed as described by Pinkel et al. (1986 (link)). Chromosomes were treated with RNAse (20 µg/mL in 2× SSC) for 1 h and with pepsin (0.005% in 10 mM HCl) for 10 min at 37 °C, followed by a step of fixation with 1% formaldehyde for 10 min and dehydration in an alcoholic series (70%/85%/100%) for 5 min. The slides were incubated in 70% formamide/2× SSC for 5 min at 72 °C and dehydrated in an alcohol series (70%/85%/100%) for 5 min. The hybridization process was performed for 16 h at 37 °C using a hybridization solution of 50% formamide, 2× SSC, 10% dextran sulfate, and denatured probe (5 ng/µL) in a final volume of 30 µL. Post-hybridization washes were performed in 15% formamide/0.2× SSC for 20 min at 42 °C, followed by washes in 0.1× SSC for 15 min at 60 °C and in Tween-20 0.5%/4× SSC for 5 min at 25 °C. Subsequently, the slides were incubated for 15 min in 5% non-fat dry milk (NFDM)/4× SSC blocking buffer and washed in 0.5% Tween-20/4× SSC for 15 min. The hybridization signals were detected using a streptavidin-FITC conjugate for the 5S rDNA probe and anti-digoxigenin rhodamine conjugate (Roche Mannheim, Germany) for the 18S rDNA probe. Chromosomes were counterstained with Vectashield/DAPI (1.5 µg/mL) (Roche Mannheim, Germany).