Chromosome Preparation
Young inflorescences were fixed in freshly prepared fixative overnight (3:1 ethanol to acetic acid), transferred to 70% ethanol and stored at -20 °C. Chromosome spreads were prepared from fixed young flower buds containing immature anthers as previously described (Mandáková & Lysak, 2016b). Chromosome preparations were treated with 100 µg/mL RNase in 2× sodium saline citrate (SSC; 20× SSC: 3 M sodium chloride, 300 mM trisodium citrate, pH 7.0) and 0.1 mg/mL pepsin in 0.01 M HCl at 37 °C for 60 min and 5 min, respectively. The preparation was then post-fixed in 4% formaldehyde in distilled water and dehydrated by passaging through increasingly pure ethanol (70%, 90% and 100%, 2 min each).