Small RNA-Directed Epigenetic Natural Variation in Arabidopsis thaliana Jixian Zhai1,2,3, Jun Liu4, Bin Liu1,2,3, Pingchuan Li1,2,3, Blake C. Meyers5,6, Xuemei Chen4, Xiaofeng Cao1* 1 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, 2 National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, 3 Graduate University of the Chinese Academy of Sciences, Beijing, China, 4 Department of Botany and Plant Sciences, Institute of Integrative Genome Research, University of California Riverside, Riverside, California, United States of America, 5 Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States of America, 6 Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, United States of America Abstract Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of ,24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of ,24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes. Citation: Zhai J, Liu J, Liu B, Li P, Meyers BC, et al. (2008) Small RNA-Directed Epigenetic Natural Variation in Arabidopsis thaliana. PLoS Genet 4(4): e1000056. doi:10.1371/journal.pgen.1000056 Editor: Joseph R. Ecker, The Salk Institute for Biological Studies, United States of America Received September 11, 2007; Accepted March 19, 2008; Published April 18, 2008 Copyright: 2008 Zhai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Basic Research Program of China (grant no. 2005CB522400), by Chinese Academy of Sciences (grant no. CXTD- S2005-2) to X.C, National Natural Science Foundation of China (grant nos. 30325015, 30430410 and 30621001) to X.C; the Meyers lab is supported by awards from the US National Science Foundation Plant Genome Research Program. Competing Interests: The authors have declared that no competing interests exist. * E-mail: xfcao@genetics.ac.cn Introduction Epigenetics, defined as the study of heritable alteration in gene expression without changes in DNA sequence, has greatly expanded our understanding of inheritance [1]. A recent study of DNA methylation by tiling array analysis of Arabidopsis Chromosome 4 in Col and Ler showed that although transposable elements (TEs) are often methylated, the methylation in the transcribed regions of genes is highly polymorphic between these two ecotypes [2]. Although epigenetic differences could potentially contribute to evolution [3–5], studies of evolution and natural variation have still been focused mainly on sequence variation, and little is known about the role of epigenetic machinery in these processes. This is primarily due to the lack of evidence for epigenetic natural variation between populations. Small interfering RNAs (siRNAs), as a key player in the epigenetic machinery, have been well documented for their general role in gene silencing at both the transcriptional and post- transcriptional levels [6,7]. In Arabidopsis, ,24 nt siRNAs can direct DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci [8]. In the RdDM process, ,24 nt siRNAs are incorporated into ARGONAUTE 4 (AGO4)- containing complexes and further guide the DOMAINS REAR- RANGED METHYLTRANSFERASE 2 (DRM2) to de novo methylate their target DNA [9,10]; once established, the non-CG methylation could be maintained by DRM2 and/or CHROMO- METHYLASE 3 (CMT3) in a locus-specific manner, and the CG methylation by METHYLTRANSFERASE 1 (MET1) [11]. Recent advances in high-throughput sequencing techniques have enabled the thorough exploration of the small RNAs populations [12–16]. Therefore, together with the complete genome sequence, we are able to directly examine whether there are regions specifically matched by siRNAs that differ among ecotypes, a situation that could lead to epigenetic natural variation. FLC, a MADS box transcription factor, is a major repressor of the transition to flowering in Arabidopsis, and many genes coordinately function in flowering time control by regulating the amount of FLC transcript [17]. In addition, allelic variation at FLC, both genetic [18–21] and epigenetic [22,23], contributes to the differences in flowering time and vernalization response among accessions, which makes FLC a classic locus for the study of natural variation in Arabidopsis. Previous studies have shown that in PLoS Genetics | www.plosgenetics.org 1 April 2008 | Volume 4 | Issue 4 | e1000056