Start Date: Aug 21, 2008
End Date: Jul 31, 2013
Slide preparation: Roots of potato allopolyploids will be collected from greenhouse-grown plants and pretreated in 0,002M 8-hydroxyquinoline at 20°C for 3 hrs. Root tips and flower buds will be fixed in a 3:1 (100% ethanol : glacial acetic acid) solution and stored in freezer (-20°C) until use. Root tips will be digested by 4% cellulase and 1% pectinase at 37°C for 80 min. The macerated root tips will be suspended by forceps in a drop of 45% acetic acid and squashed. Slides will be pretreated by pepsin solution (final concentration 0.1 mg/ml) for 45 min at 37°C and subsequently incubated in a RNase A solution (6 'l stock solution – 10 'g/'l + 24 'l 2xSSC per slide; 40 min at 37'C) and then – in formaldehyde solution (4% for 10 min). After each treatment slides will be washed in 2x SSC buffer 5 min for three times at room temperature. Finally slides will be incubated in 70%, 90%, 100% ethanol series, for 3 min each at room temperature. DNA extraction and labeling: Genomic DNA will be isolated from the putative diploid progenitor species of the allopolyploids as determined by results from Spooner’s DNA phylogenetic studies using young leaves of greenhouse-grown plants. The GISH technique will be done according to published protocols with minor modifications. DNA will be either labeled with DIG-UTP or Biotin-UTP by nick-translation (DIG- and Biotin-Nick Translation Mix, Roche, cat. No. 11745816910, cat. No. 11745824910). Hybridization: Hybridization mix (40 'l per slide) for GISH will be prepared with differential labeled DNA from the putative parental species and included: sheared fish sperm DNA (20 µg), Probe DNA of one parent (100 ng), Probe DNA of the other parent (100 ng), 10% dextran sulfate, deionized formamide (50%). Hybridization will be performed over night at 37'C. Detection: DIG-labeled DNA will be detected with rhodamine anti-DIG conjugate and biotin labeled probes detected with FITC conjugated avidin (Roche, cat. No. 11207750910, cat. No. 11975595910). 29 'l of blocking reagent (30 mg BSA solution in 999 'l 4x SSC) will be added to slides, followed by incubation for 30 min. at room temperature. The antibody solution composed of 1 'l Anti-DIG-rhodamine stock solution + 1 'l Avidin- fluorescein stock solution + 28 'l Detection buffer (DB: 0.1 g BSA dissolved in 9.9 ml 4 x SSC, pH=7.4) will be added to each slide; incubation for 45 min at 37'C. Slides will be washed three times in 4x SSC (pH=7,4) (5 min each) at 42'C. Chromosomes will be counterstained by 4’, 6-diamidino-2-phenylindole (DAPI) in Vectashield antifade solution (Vector Laboratories). All images will be captured digitally using a SenSys CCD (charge coupled device) camera (Roper Scientific, Tucson, AZ) attached to an Olympus BX60 epifluorescence microscope. The CCD camera will be controlled using IPLab Spectrum v3.1 software (Signal Analytics, Vienna, VA) on a Macintosh computer.