Axioscope a1 imager fluorescent microscope

Chromosome preparation, chromosome spreading and GISH experiments were performed as described in D’hont et al. [24] (link). Genomic DNA from E. arundinaceus HN 92-77 and HN 92–105 was labeled with digoxigenin-11-dUTP (Roche) and genomic DNA from Badila and CP 84–1198 was labeled with biotin-16-dUTP (Roche) using the Nick Translation Kit (Roche). To detect signal from biotin-labeled probes, Avidin D, Rhodamine 600 (XRITC) and biotinylated anti-avidin antibody (Vector Laboratories, Burlingame, CA) were used. To detect signal from digoxigenin-labeled probes, sheep-anti-digoxin-FITC (Roche, Lewes, UK) and rabbit-anti-sheep-FITC (Roche, Lewes, UK) were used. Chromosomes were then counter stained using DAPI in Vectashield anti-fade solution Vectashield (Vector Laboratories, Burlingame, CA). The hybridization signals were observed on an AxioScope A1 Imager fluorescent microscope (Carl Zeiss, Gottingen, Germany). Images were captured digitally with an AxioCam MRc5 and AxioVision v.4.7 imaging software (Carl Zeiss, Gottingen, Germany).

GISH technique were performed as described previously by D’Hont et al. [32 (link)] with moderate improvement. The denaturing solution included 70% formamide in 2× SSC. Slides were denatured in this solution for 3 min at 80 °C. Dehydration was performed in cold ethanol and slides were then air dried at room temperature. The probe mixture including hybridization buffer (50% formamide, 2× SSC, 10% dextransulfate) and 200 ng labeled probe after denaturation for 10 min at 97 °C was applied to each slide and incubated for 20 h at 37 °C in a humid dark box. The high stringency conditions of post-hybridization washes were carried out with 2 × SSC for 8 min at 42 °C, a second wash in 50% formamide, 2 × SSC, pH 7.0, for 3 × 8 min at 42 °C, followed by a rinse in 2 × SSC for 8 min at room temperature and a final wash in 0.1 × SSC for 3 × 8 min at 55 °C. The biotin-labelled probe was detected with avidin-conjugated Texas red and the digoxigenin-labelled probe was detected with FITC (fluorescein isothiocyanate)-conjugated anti-digoxigenin antibody. Slides then were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) in a Vectashield anti-fade solution (Vector Laboratories, Burlingame, CA). GISH signals were captured using the AxioVision measurement module of AxioScope A1 Imager fluorescent microscope (Zeiss, Germany).

Chromosome preparation and the GISH experiment were carried out according to the method described by D’hont et al. [9 (link)]. Genomic DNA from Badila (S. officinarum) and YN82-114 (S. spontaneum) was labelled with Biotin, the Biotin-labeled probe was detected with Avidin D, Rhodamine 600 (XRITC) and a Biotinylated anti-avidin antibody (Vector Laboratories, Burlingame, CA), respectively. Genomic DNA from HN92-77 or HN92-105 (E. arundinaceus) was labelled with Digoxigenin, and the Digoxigenin-labeled probe was detected with sheep-anti-Digoxin-FITC (Roche, Lewes, UK) and rabbit-anti-sheep-FITC secondary antibody (Roche, Lewes, UK). Chromosomes were then counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) in a Vectashield anti-fade solution (Vector Laboratories, Burlingame, CA). FISH signals were captured using an AxioScope A1 Imager fluorescent microscope (Carl Zeiss, Gottingen, Germany). In this study, results are presented as the modal number and the range of chromosomes counting four to 22 metaphases for each progeny (Table 2). The images were processed using an AxioCam MRc5 and AxioVision v.4.7 imaging software (Carl Zeiss, Gottingen, Germany).