Holoploid genome size (Greilhuber et al. 2005) was estimated by means of propidium iodide FCM. For each plant, a piece of intact tissue (usually leaf or stem or ovary) was chopped together with an appropriate amount of an internal reference standard using a new razor blade in a Petri dish containing 0.5 ml of ice-cold Otto I buffer (0.1 M citric acid, 0.5% Tween 20) (Otto 1990, Doležel et al. 2007). The resulting suspension was filtered through a 42-µm nylon mesh and incubated at room temperature for at least 5 min. After incubation, the suspension was stained by using 1 ml of Otto II buffer (0.4 M Na2HPO4 · 12 H20) supplemented with the intercalating fluorescent dye propidium iodide, RNAase IIA (both at final concentrations of 50 µg/ml) and β-mercaptoethanol (2 µl/ml). Samples were stained for 5 min at room temperature and analysed using a Partec CyFlow cytometer (Partec GmbH., Münster, Germany) equipped with a 532 nm diode-pumped solid-state laser Cobolt Samba (Cobolt AB, Solna, Sweden) as the source of excitation light. Fluorescence intensity of 5000 particles was recorded, and data were analysed using Partec FloMax Software version 2.4d. Pisum sativum ‘Ctirad’ (2C = 8.76 pg; Doležel et al. 2007) served as the primary reference standard. Additional secondary standards (see table below) were used either to avoid a large difference in genome size between the standard and examined sample, or if the primary standard was unavailable. Nuclear genome sizes of all additional standards were recalibrated using Pisum sativum, based on repeated measurements on different days.
Additionally, AT-selective DAPI dye was used for the estimation of base composition. Sample preparation was exactly the same, but propidium iodide and RNAase were supplemented by DAPI dye at a final concentration 4 µg/ml. Analyses were conducted on a Partec Space cytometer (Partec GmbH., Münster, Germany) equipped with UV-led chip (365 nm). Computation of the base content followed Šmarda et al. (2008) via a publicly available excel spreadsheet.