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).