Small RNA-Directed Epigenetic Natural Variation Figure 3. Methylation Analysis of MPF and FLC-TE. Bisulfite sequencing analysis of DNA methylation at the MPF (A), FLC-TE (B) and AtSN1 (C) in Col, Ler, hen1-1, ago4-1, kyp-2, drm2 56Ler, and cmt3-7, summarized in different sequence contexts. Methylation status had been independently confirmed by bisulfite sequencing or McrBC PCR for at least four times. doi:10.1371/journal.pgen.1000056.g003 Methylation at MPF Is Independent of the TE Insertion Nearby Since MPF is methylated and it is near to the TE insertion in FLC-Ler, it was of interest to investigate whether the methylation at MPF is induced by the TE. We examined the methylation status of MPF in several accessions that are also reported to contain transposable elements inserted in the first intron of FLC (Figure S3A) [19,20]. These were tested by McrBC-PCR [34] (for Bd-0, JI-1, Stw-0, Kin-0 (CS1273), and Gr-3) and bisulfite sequencing (for Da(1)-12). Although the MPF is methylated in Bd-0, JI-1 and Kin-0 (CS1273), it remains unmethylated in Stw-0, Gr-3 and Da(1)-12 (Figure S3B, and data not shown for Da(1)-12) indicating that the TE insertions nearby are dispensable for the methylation at MPF. A previous study using 27 Arabidopsis accessions showed that the FLC-TE in Ler was also detected in Dijon-G and Di-2 (Figure S3A) but was absent in the closely related Landsberg-0 or Di-1 [18]. McrBC-PCR analysis showed that MPF is methylated in all four of these accessions, even in those without the FLC-TE insertion (Figure S3C), which further confirmed that the methylation at MPF is independent of the TE insertion nearby. Origin of MPF-siRNAs To study the origin of the MPF-siRNAs, we found that a 220 bp sequence at MPF is absent in one Kin-0 accession (CS6755, different from the Kin-0 (CS1273) accession mentioned above that contains a methylated MPF). Further analysis revealed that this difference is caused by the insertion of a non-autonomous hAT element [35] with the typical 8 bp TSD (target site duplication) and short terminal inverted repeats (TIRs) (Figure 4 and Figure S1). However, MPF-siRNAs in Ler are probably not derived from other hAT elements because those MPF-siRNAs with the full length information from 454 sequencing in Ler [15] have only one match (at MPF) in the genome; also, genomic Southern blot hybridization revealed that Ler do not contains extra copy of this hAT element comparing to Col (Figure S4). Therefore, the MPF-siRNAs are probably generated from MPF itself. Methylation State at MPF in Ler Is Transmissible to Col by Genetic Crossing but with Extensive Diversity in the F1 In paramutation, the silenced paramutagenic lines are able to confer the active state of the paramutable lines, and make them become paramutagenic [36]. To test whether the methylated state at MPF in Ler is transmissible, we performed bisulfite sequencing to investigate the DNA methylation status in four F1 lines from the crosses of both Col R6Ler = and Ler R6Col =, with the single nucleotide polymorphisms (SNPs) at MPF (Figure S1) used to distinguish the Col and Ler derived sequencing results (Figure 5A). In addition, twenty-four more lines from reciprocal crosses were tested for their MPF methylation by real-time McrBC-PCR (Figure 5B). These experiments revealed extensive diversity in the methylation status of MPF in each individual line in the F1 generation. This diversity could be summarized in the following way: 1) in some lines, the MPF-siRNAs from Ler are able to trigger the de novo methylation at Col-derived MPF; 2) in some other lines, not only the Col-derived MPF remains unmethylated, the Ler-derived MPF could even lose its methyl- ation; 3) there are also cases in which the Ler-derived MPF remains methylated and Col-derived MPF remains unmethylated, just like their ancestors; therefore the MPF is semi-methylated in the whole plant. Figure 4. Structure of MPF in Three Accessions. Ler and Col contain a 220 bp hAT element insertion (gray box); both the insertion and the target site duplication are absent in Kin-0. The MPF-siRNAs precisely match to one end of this insertion at both strands. 17 nt siRNA tags are from Col-derived MPSS dataset; the rest 22,24 nt siRNAs sequences are from Ler-derived 454 dataset. doi:10.1371/journal.pgen.1000056.g004 PLoS Genetics | www.plosgenetics.org 4 April 2008 | Volume 4 | Issue 4 | e1000056