A simple flow cytometry-based assay to study global methylation levels in onion, a non-model species.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01047-6. © Prof. H.S. Srivastava Foundation for Science and Society 2021.