Author Summary Phenotypic variation has been mainly attributed to their differences in genetic materials, i.e., the DNA sequence. The advances in Epigenetics in past decades has revealed it as a fundamental mechanism that could inheritably influence gene function without change in DNA sequence, but by modulating chemical modifications on DNA itself (methylation), or on histone proteins, which package the DNA further into nucleosome. Nevertheless, the roles of epigenetic regulation in natural variation were not explored much because of the limitation in high-through- put analytical tools. A recent study in model plant Arabidopsis showed that there are many DNA methylation polymorphisms between the two ecotypes. In plant, a subset of RNA named small interfering RNA (siRNA), is capable of triggering the epigenetic modifications on DNA or histone at their target region with complementary nucleotide sequences. Here, we took a view from the small RNA side and by applying molecular and bioinformatic approaches we showed that the same region could be led to different epigenetic status because of the difference in their corresponding small RNA abundance and between the two closely related Arabidopsis ecotypes, suggesting that there could be small RNA-directed epigenetic differences among natural populations. Ler, a 1224 base pair (bp) nonautonomous Mutator-like transpos- able element (TE) inserted in the first intron of FLC (FLC-TE-Ler) [19] was methylated and heterochromatic under the direction of ,24 nt siRNAs generated by homologous TEs, and mutation of HUA ENHANCER 1 (HEN1) in Ler (hen1-1), a key component in small RNA biogenesis [7], released the transcriptional silencing of FLC-Ler [22]. In this study, we discovered a cluster of ,24 nt siRNAs that are present at high levels in the ecotype Ler and that could direct DNA methylation and heterochromatinization adjacent to FLC promot- er [24]. However siRNAs matching to the same region in Col are of low abundance and cannot direct DNA methylation. Further- more, from comparisons between Ler and Col of small RNA data produced by high-throughput sequencing, we identified at least 68 loci that are matched by significant levels of ,24 nt siRNAs, and 88% are methylated in Ler but not Col from a set of 42 loci that were examined.. Although siRNA clusters are often heavily methylated [25] and a large proportion of the methylation polymorphisms between Col and Ler are not associated with small RNAs [2], our data reveal that there could still be considerable small RNA-directed epigenetic natural variation between two ecotypes of Arabidopsis. Results A Region Adjacent to the Promoter of FLC is Methylated in Ler but not Col In addition to the previously described Mutator-like transposable element (TE) inserted in the first intron of FLC [19] in Ler, we found that a region located adjacent to the promoter of the FLC was specifically methylated in Ler but not in Col (Figure 1A). We named this region MPF (Methylated region near Promoter of FLC). Restriction enzymes including AciI, HpyCH4 IV and Fnu4HI, which are sensitive to CpG methylation, were able to cut outside of the MPF but not within this region in Ler (Figure 1). Notably different from the TE inserted in FLC-Ler, the MPF of Ler and Col share almost identical sequences (Figure S1). Bisulfite sequencing of MPF (B1 region, Figure 2A) revealed that a small region of less Small RNA-Directed Epigenetic Natural Variation Figure 1. DNA Methylation Analysis of the FLC Promoter by Southern Blots. (A) A diagram of the genomic region around FLC promoter is shown above with the positions of restriction sites marked as follows: Fnu4H I (F), Aci I (A), and HpyCH4 IV (H) are sensitive to methylation; Nde I (N) which is not sensitive to methylation is used as a negative control. Red stars highlight the methylated sites. The digested fragments that could be detected by probe covering FLC promoter (gray strip) were diagramed and the size is indicated by numbers (in kilobases) beneath the fragments. A hAT element is represented as gray box (see Figure 4 for more detail). (B) Determination of DNA methylation status at FLC promoter in Ler, hen1-1, Col, and hen1-4. Black arrows indicate the DNA fragments which contain the methylated (and therefore uncut) enzyme recognition sites. doi:10.1371/journal.pgen.1000056.g001 than 100 bp was exhibited a very high level of asymmetric methylation (also called CHH methylation, where H represents A, C or T) (Figure 2C). This region also demonstrated extensive CpG and CNG (where N is any nucleotide) methylation (Figure 2C). In addition, no DNA methylation was found outside the MPF (the B2 and B3 regions, Figure 2A) in Ler (data not shown) or the MPF in Col (Figure 3A) by bisulfite sequencing. High Levels of MPF-siRNAs in Ler, but not Low Levels in Col, Direct DNA Methylation and Heterochromatinization at MPF Since asymmetric methylation is the hallmark of RdDM [26], we decided to verify whether there are corresponding siRNAs PLoS Genetics | www.plosgenetics.org 2 April 2008 | Volume 4 | Issue 4 | e1000056