Small RNA-Directed Epigenetic Natural Variation Figure 7. DNA Methylation Analysis of 44 loci Varied in Small RNA Abundance between Col and Ler using Real-time McrBC-PCR. (A) Real-time PCR results using McrBC non-digested (white bar) and digested (black bar) DNA from both Col (under the axis) and Ler (above the axis) as the PCR templates. For the comparison, the Non-digested result of each locus was normalized to 1. means PCR amplification failed. If the value of McrBC digested sample at certain locus is significantly lower than McrBC non-digested one, then this locus is methylated, otherwise it is unmethylated. Locus #60 which is methylated in both Col and Ler is used as the positive control and unmethylated Actin is used as the negative control. (B) Summary of the McrBC results. ‘‘Methylated’’ is defined as the value of McrBC non-digested sample at a certain locus is lower than 0.5, and ‘‘unmethylated’’ is defined as the value of McrBC non-digested sample at a certain locus is higher than 0.5. doi:10.1371/journal.pgen.1000056.g007 which the DNA or chromatin are open to modifiers like the MPF- siRNA containing RISC (RNA induced silencing complex) from Ler. However, this open stage must be very short, and when it is over, the epigenetic state, no matter active or silenced, will be maintained in the following developmental processes, so that the unmethylated state of Col-derived MPF and the methylated state of Ler-derived MPF could well maintained in Ler R6Col =line #2 (Figure S5A). Thus far, the function of ,24 nt siRNAs in plants has mainly been ascribed a role in silencing transposable elements and repeat- associated sequences [39]. Thus, it is unclear how Ler and Col, both with the functional RNAi machinery, might acquire many siRNA-directed epigenetically variable loci. One characteristic of MPF-siRNAs, their very restricted location (all matching to a region less than 50 bp), may confer on them more flexibility than other, larger silent loci. PLoS Genetics | www.plosgenetics.org 8 April 2008 | Volume 4 | Issue 4 | e1000056