Bisulfite sequencing for cytosine-methylation analysis in plants

RNA silencing is a sequence-specific RNA degradation mechanism conserved in eukaryotes including fungi, plants, and animals. One of the three RNA silencing pathways is DNA methylation which is the result of interaction between DNA and siRNA, a hallmark of RNA silencing. Bisulfite sequencing can be very powerful for DNA methylation analysis in this context. This method includes DNA extraction, digestion of DNA with restriction enzyme, treatment of DNA with bisulfite, PCR amplification of DNA, cloning of amplified DNA fragments, sequencing of DNA fragments, and analysis of DNA sequences. Based on this method, increased levels of cytosine methylation were obtained in both symmetrical (CpG, CpNpG) and non-symmetrical (CpHpH) contexts in silenced lines of transgenic plants (CG>CNG>CHH), while the methylation levels were low in nonsilenced, over-expressing lines. Through grafting, RNA silencing was induced in the non-silenced scions from silenced rootstocks; however, the methylation level of DNA in the scions did not increase.