Cytoexpert v2

The total nuclear DNA content (2C-DNA value) of the seven Senna species were measured using flow cytometry by following the method outlined by Bourge et al. (2018) (link). Briefly, fresh leaves were collected from ten individuals of each species and mixed with an internal reference standard (Dendropanax morbifera, 2C DNA = 4.09 pg). In a petri dish, the samples were treated with the isolation LB01 buffer and passed through two nylon mesh filters (CellTrics Filters, Sysmex Asia Pacific, Singapore) with pore sizes of 50 µm and 20 µm, respectively. The filtered nuclei were stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA, cat. no. P4170; Molecular Probes; cat. no. P3566) and RNase A (Sigma-Aldrich, St. Louis, MO, USA, cat. no. R5000) and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). The DNA content of the samples was computed by analyzing the peaks of the standards and samples using CytoExpert v2.3 software (Beckman Coulter Inc., Pasadena, CA, USA). The 2C values were calculated by estimating the linear fluorescence intensity of the stained nuclei for each species and the internal standard. The relative genome size was determined using a formula following Nguyen et al. (2023) (link). The coefficient of variation (CV) was below 3% on average and did not exceed 7%.