DNA methylation is an important epigenetic mark and global methylation dynamics regulate plant developmental processes. Even though genome sequencing technologies have made DNA methylation studies easier, it is difficult in non-model species where genome information is not available. Therefore in this study, we developed a simple assay for analysing global methylation levels in plants by washless immunolabelling of unfixed nuclei using flow cytometry. Onion leaf tissue was used as a model system, and mean fluorescence intensity due to anti-5- methyl cytosine (5-mC) antibodies were used as a measure of global methylation levels. Among three nuclear isolation buffers evaluated, the highest nuclear yield with the low background was obtained with LB01. To maintain a balance between high DNA fluorescence value and low coefficient of variation of DNA peaks, 45 min of hydrolysis with 0.2 N hydrochloric acid was used for chromatin denaturation resulting in six-fold increase in 5-mC fluorescence compared to control. This method was used successfully to detect 5-Azacytidine induced DNA hypomethylation in onion leaf tissues.

DNA甲基化是一个重要的表观遗传标记，全球甲基化动态调节植物发育过程。尽管基因组测序技术使 DNA 甲基化研究变得更容易，但在基因组信息不可用的非模型物种中却很困难。因此，在本研究中，我们开发了一种简单的测定方法，通过使用流式细胞仪对未固定的细胞核进行免洗免疫标记来分析植物中的全局甲基化水平。洋葱叶组织用作模型系统，由抗 5-甲基胞嘧啶 (5-mC) 抗体引起的平均荧光强度用作整体甲基化水平的量度。在评估的三种核分离缓冲液中，LB01 获得了具有低背景的最高核产量。为了保持高 DNA 荧光值和低 DNA 峰变异系数之间的平衡，使用 0.2 N 盐酸水解 45 分钟进行染色质变性，导致 5-mC 荧光比对照增加六倍。该方法成功用于检测洋葱叶组织中 5-氮杂胞苷诱导的 DNA 低甲基化。