Facs software 1

Fully expanded leaf tissue from each sample collected in the field was dried in silica gel. Approximately 1 cm 2 of the sample was chopped along with the internal standard [Oryza sativa subsp. japonica S. Kato, 1930 'Nipponbare', 2C = 0.91 pg, (Uozu et al. 1997) (link) ] using a sharp razor blade in a Petri dish containing 1 ml of 'woody plant buffer' (WPB, Loureiro et al. 2007 (link)), following the one-step procedure proposed by Doležel et al. (2007) (link). The nuclear suspension was then filtered through a nylon mesh (400 µm) to remove debris and stained with 50 µl PI. After incubation for 10 min on ice, the relative nuclear DNA content was estimated by recording at least 3000 particles using a BD Influx flow cytometer fitted with a blue laser (488 nm, 200 mW) and analysing three replicates of each individual. The resulting histograms were analysed with the BD FACS software 1.0.0.650. The 2C-value was calculated using the linear relationship between fluorescence signals from stained nuclei of the unknown sample and the internal standard. 1Cx was calculated dividing the 2C-value by the ploidy.