Anti digoxigenin fitc

Total H. chilense genomic DNA was also labelled by nick-translation with biotin-11-dUTP (Boehringer Mannheim Biochemicals, Germany) or digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN, USA) and used as a probe. The in situ hybridisation protocol was performed according to Prieto et al. (2004b (link)). The amount of either the biotin- or digoxigenin-labelled probes in the hybridisation mixture was 5 ng. Unlabelled wheat genomic DNA was used as blocking DNA at a ratio of 1:50 (probe/blocking DNA). Biotin-labelled H. chilense DNA and digoxigenin-labelled H. chilense DNA were detected with a streptavidin-Cy3 conjugate (Sigma, St. Louis, MO, USA) and antidigoxigenin-FITC (Roche Diagnostics, Meylan, France), respectively. Chromosomes were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). Hybridisation signals were visualised using a Nikon Eclipse 80i epifluorescence microscope. Images were captured with a Nikon CCD camera using the Nikon 3.0 software (Nikon Instruments Europe BV, Amstelveen, The Netherlands) and processed with Photoshop 4.0 software (Adobe Systems Inc., San Jose, CA, USA).

Chromosome preparations, DNA fibers obtention, single and double-color FISH, and Fiber-FISH experiments were conducted as described in Kuhn et al. (2008) (link). The probes labeled with digoxigenin-11-dUTP were detected with antidigoxigenin FITC (Roche) and probes labeled with biotin-14-dATP were detected with NeutrAvidin-rhodamine (Roche). Chromosomes were stained with DAPI (4′, 6-diamidino-2-phenylindole, dihydrochloride salt). The preparations were analyzed under an epifluorescence Zeiss Axiophot 2 microscope equipped with a CCD camera and the images were obtained using the AxioVision software (Zeiss). To determine the size of the DNA fibers, hybridization signals were measured according to the protocol described by Schwarzacher and Heslop-Harrison (2000) .

The FISH method was conducted as follows: slides with fixed chromosomes were maintained at 37 °C for 1 h. Subsequently, they were incubated with RNAse (10 mg/ml) for 1 h at 37 °C in a moist chamber. Next, it was performed a 5-min wash with 1xPBS and 0.005 % pepsin was applied to the slides (10 min at room temperature). The slides were then washed again with 1xPBS. The material was fixed with 1 % formaldehyde at room temperature for 10 min. After further washing, the slides were dehydrated with 70, 85 and 100 % ethanol, 2 min in each bath. The chromosomal DNA was denatured in 70 % formamide/2xSSC for 3 min at 72 °C. The slides were dehydrated again in a cold ethanol series (70, 85 and 100 %), 5 min each. The hybridization mixture, containing 100 ng of denatured probe, 10 mg/ml dextran sulfate, 2xSSC and 50 % formamide (final volume of 30 μl) were heated to95 °C for 10 min and then applied on the slides. Hybridization was performed for a period of 16–18 h at 37 °C in a moist chamber. After hybridization, the slides were washed for 5 min with 2xSSC and then rinsed quickly in 1xPBS. The detection of the probes was performed with Streptavidin-Cy3 (Sigma) for the 18S rDNA probe and anti-digoxigenin-FITC (Roche) for the 5S rDNA probe. The chromosomes were counterstained with DAPI (1.2 g/ml) in Antifading solution (Vector Laboratories).