2.2 Genome size estimation by flow cytometry
Flow cytometry was used to estimate the genome size of C. japonica according to the standard procedure (He et al. , 2016). One head of male adult was homogenized completely with 500 μL ice-cold Galbraith’s buffer (PH=7). The homogenate was centrifuged at 5,000 rpm. at 25 ℃ for 5 min and suspended with 400 μL phosphate buffer (PH=7.4). To remove the RNA, RNaseA was add into tube at 25 ℃ for 10 min (final concentration of 20 ug/mL). Finally, samples were stained with 50 μg/mL propidium iodide stock solution in darkness at 4 ℃ for 10 min. Samples were analyzed by FACSCalibur platform (BD Biosciences) and FACScomp v4.0 under 488-nm wavelength. FlowJo v7.6.1 was used to obtain the nuclei peaks. Drosophila melanogaster was analyzed as a control following the above same parameter. The outputs were used to estimate the genome size.