Small RNA-Directed Epigenetic Natural Variation Figure 6. Illustration of the Strategy for Identifying Loci Matched by Significant Level of ,24 nt siRNA Specifically in Ler using Chromosome 3 as an Example. Unique small RNAs obtained by 454 sequencing from Col and Ler $23 nt were mapped to the genome, then the perfect matches were counted per 100 bp. With this information, a filter was used to further identify loci with no less than three hits within 300 bp in Ler versus no hits within 1500 bp for the same region in Col. doi:10.1371/journal.pgen.1000056.g006 complex agricultural traits have been widely used. Recent progress in epigenetics has revealed mechanisms that can heritably regulate gene function without alteration of primary nucleotide sequences. Although the importance of epigenetic natural variation have become more and more noticed [3,5], the role of epigenetic regulation in evolution has been less well studied due in part to limitations in the techniques used for the investigation of epigenetic variation among natural populations. Recently, sub- stantial improvements in high-throughput analysis approaches have made it possible for the effective detection of variation in DNA methylation, histone modifications and small RNA abun- dances [2,12–16,25,40]. Small RNAs that can target DNA methylation and chromatin modifications have been proposed as a potential source in inherited epigenetic differences [3], and the latest techniques offer rapid and relatively inexpensive means for the profiling of small RNAs. In this study, we discovered that a hAT element adjacent to the promoter of FLC, which we named MPF, is methylated and heterochromatic in Ler but not Col because of their differences in the abundance of corresponding siRNAs. Furthermore, by comparisons between Ler and Col of publicly available small RNA data produced by high-throughput sequencing [15,16], we identified at least 68 loci that are matched by significant levels of ,24 nt siRNAs, and 88% examined loci are methylated specifically in Ler but not Col. Our data reveal that there could be a considerable amount of small RNA-directed epigenetic natural variation between two ecotypes of Arabidopsis. Although we identified dozens of loci, this analysis is still far from saturating. A Sadhu element (At2g10410), which was reported to be epigenetically silenced in Ler and other 18 strains but highly expressed in Col, did not show up among the 68 loci [41]; although bisulfite sequencing revealed that this element contains CNG and asymmetric methylation in Ler, which is presumably siRNA-directed to some extent [41]. Furthermore, hundreds of additional loci with one or two hits specifically in Ler (data not shown) may also be silent; these may be better characterized when additional Ler small RNA and genome sequence data become available. Two examples of siRNA-associated, naturally-occurring epige- netic variation have been well studied in plants, including the phosphoribosylanthranilate isomerase (PAI) gene family in Arabi- dopsis and paramutation in maize [36]. In some Arabidopsis ecotypes, two PAI genes form an inverted repeats that may generate siRNAs and silence related members in the same gene family [42]. Paramutation, the allele-dependent transfer of heritable silencing state from one allele to another [36], is associated with another type of repeats, the tandem repeats. MEDIATOR OF PARAMUTATION 1 (MOP1) [43], whose deficiency disrupts paramutation, is an ortholog of the Arabidopsis RDR2 (RNA Dependent RNA polymerase 2), an essential component of RNAi machinery [6]. Notably, epigenetic variation at the MPF is quite different from these two cases: first, neither inverted- nor tandem-repeats features were found at MPF or elsewhere in the genome with similar sequence; second, the level of MPF-siRNAs is high in Ler and low in Col, instead of all-or-none; third, the restricted location of MPF-siRNAs is markedly different from the dispersed distribution of siRNAs from most inverted or tandem repeats [12]. Although paramutation phenomenon had been well document- ed, the details of how the silencing signal is transmitted from one allele to the other in the F1 heterozygote are still less understood. In our study, the diverse methylation status among individuals in F1 generation of the reciprocal crosses from Col6Ler indicate that there might be a reprogramming stage shortly after fertilization, in PLoS Genetics | www.plosgenetics.org 7 April 2008 | Volume 4 | Issue 4 | e1000056