Comparative Chromosome Painting
For comparative chromosome painting (CCP), 674 chromosome-specific BAC clones of A. thaliana (The Arabidopsis Information Resource, TAIR; http://www.arabidopsis.org) were used to establish contigs corresponding to the 22 GB and eight chromosomes of the ACK (Lysak et al., 2016). BAC-probes were labeled with biotin-dUTP, digoxigenin-dUTP or Cy3-dUTP by nick translation as previously described (Mandáková & Lysak, 2016a). DNA probes were pooled to follow the given experimental design, ethanol precipitated, dried and dissolved in 20 μL of 50% formamide and 10% dextran sulfate in 2× SSC. The 20 μL of the dissolved probe was pipetted on a chromosome-containing microscopic slide and immediately denatured on a hot plate at 80 °C for 2 min. Hybridization was carried out in a moist chamber at 37 °C overnight. Post-hybridization washing was performed in 20% formamide in 2× SSC at 42 °C. Hybridized probes were visualized either as the direct fluorescence of Cy3 or through fluorescently labeled antibodies against biotin and digoxigenin as previously described (Mandáková & Lysak, 2016a). Chromosomes were counterstained with 4´,6-diamidino-2-phenylindole (DAPI, 2 µg/mL) in Vectashield antifade. Fluorescence signals were analyzed and photographed using a Zeiss Axioimager epifluorescence microscope equipped with a CoolCube camera (MetaSystems). Images were acquired separately for all four fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik). The four monochromatic images were pseudocolored, merged and cropped using Photoshop CS (Adobe Systems) and ImageJ (National Institutes of Health).