Biotin 11 dutp

For the preparation of the genomic probe, total genomic DNA was extracted from young leaves of subsp. sativa using the Qiagen mini kit (Qiagen Ltd., Manchester, UK) according to the manufacturer’s instructions. Then, DNAs were labelled with biotin-11-dUTP (Sigma Aldrich Co., Steinheim, Germany) or digoxigenin-11-dUTP (Roche, Indianapolis, IN, USA) by nick translation. The plasmid pUc18 containing the sequences (TTTAGGG)n of Arabidopsis thaliana was labelled with biotin-11-dUTP by PCR for identification of the telomere repeat sequences (TRSs). Clone pTa71 containing a 9-kb EcoRI fragment of the 18S-5.8S-25S rRNA genes and the intergenic spacers from wheat [18 (link)] was labelled with digoxigenin-11-dUTP and used to localize the sites of 45S rDNA.

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).

The 5S and 45S rDNA loci were localised using probes pA5S, pA18S and pA26S isolated from genomic DNA of Arachishypogaea (Robledo and Seijo 2008 ) and labelled by nick translation with digoxigenin-11-dUTP (Roche Diagnostics) or biotin-11-dUTP (Sigma-Aldrich). Pretreatment of slides, chromosome and probe denaturation, conditions for the in situ hybridisation (hybridisation mixes contained DNA probes at a concentration of 2.5–3.5 ng/µl, with a stringency to allow sequences with 80%–85% identity to remain hybridized), post-hybridization washing, blocking and indirect detection with fluorochrome-conjugated antibodies were performed according to Seijo et al. (2004) . The first set of antibodies consisted of anti-biotin produced in goat (Sigma-Aldrich) and monoclonal anti-digoxigenin conjugated to fluorescein isothiocyanate (FITC) produced in mouse (Sigma-Aldrich). The second set consisted of anti-goat conjugated to tetramethyl-rodamine isothiocyanate (TRITC) produced in rabbit (Sigma-Aldrich) and anti-mouse conjugated to FITC produced in sheep (Sigma-Aldrich). Preparations were counterstained by mounting them with Vectashield medium (Vector Laboratories) containing 2 mg/ml of 4',6-diamidino-2-phenylindole (DAPI).