Genomic in situ hybridization

More than 15 samples were examined by GISH analysis. Genomic DNA was extracted from young leaves of the two parents and the putative hybrids using the CTAB method [67]. DNA was then labeled with DIG-11-dUTP (Roche Ltd.) via nick translation and post-fixed in 4% paraformaldehyde for 10 min. The chromosomal DNA was denatured in 70% formamide in 2× SSC at 70°C for 2.5 min and dehydrated through an ethanol series at 4°C. The hybridization mixture consisted of 50% formamide, 10% dextran sulfate, 2× SSC, 0.1% SDS, and 50 ng/μl of DNA probe. Hybridization was performed at 37°C overnight. The slides were washed in 20% formamide in 0.1× SSC at 42°C for 10 min, in 2× SSC at 42°C for 10 min, and in 2× SSC at room temperature for 5 min. The labeled probe was detected with fluorescein-conjugated antibodies (Roche Molecular Biochemicals), and the chromosomes were counterstained with propidium iodide (PI). The prepared materials were observed by confocal fluorescence microscopy (Leica CTR 6500). Furthermore, the GISH analysis in this study did not use the blocking DNA, since chromosomes derived from the female parent, A. John De Biase ‘Blue’, or from the male parent, P. Chih Shang's Stripes, can be clearly distinguished without the use of blocking DNA.