Estimating DNA Content in Grape Varieties

DNA content was estimated using flow cytometry as described in Cochetel et al. [47 (link)]. The nuclei extraction was performed using the Cystain PI absolute P kit (Sysmex America Inc., IL, USA). For the internal reference standard, Lycopersicon esculentum cv. Stupické polní tyčkové rané was selected, with a known genome size of 2 C = 1.96 pg; 1 C = 958 Mb [73 (link)]. Young leaves (~ 5 mg) from grape and tomato were finely cut with a razor blade in a Petri dish containing 500 mL of extraction buffer and filtered through a 50-mm filter (CellTrics, Sysmex America Inc., IL, USA). Propidium iodide staining solution (2 mL) was then added to the nuclei suspension [74 (link), 75 (link)]. Measurements were acquired with a Becton Dickinson FACScan (Franklin Lakes, New Jersey) equipped with a 488-nm laser. Raw data were imported and analyzed with the R package flowPloidy v.1.22.0 [76 (link)] (Additional File 1: Table S9). For V. berlandieri, V. riparia, and V. rupestris, estimates from the same species were retrieved from literature [77 (link)].