Genetic variability (due to insertion, deletion and point mutation) occurs stochastically, at very low frequency, primarily irreversibly and is often recessive. In contrast, heritable epigenetic variability may be more appropriate to regulate, rather than disrupt or create, gene function, and thus may be an ideal or more dynamic force for evolutionary change of gene regulation. Materials and Methods Plant Materials The Bd-0 (CS962), JI-1 (CS1248), Stw-0 (CS1538), Gr-3 (CS1202), Kin-0 (CS1273, CS6755), Da(1)-12 (CS917), Dijon-G (CS910), Di-1 (CS1108), Di-2 (CS1110), and La-0 (CS1299) accessions of Arabidopsis were acquired from ABRC; hen1-1 (Ler background), hen1-4 (Col background), and dcl1-9 mutants were described before [22]; cmt3-7, kyp-2, ago4-1, and drm2 56Ler were generous gifts from Steve Jacobsen at UCLA. The AGO4 complementation lines were kindly provided by Gregory J. Hannon at CSHL and Yijun Qi at NIBS. Small RNA Northern Blot RNAs were extracted from 20-day-old, soil-grown plants. 32P end-labeled LNA probe was used for hybridization. Total RNAs were extracted using Trizol solution (Invitrogen) from 20-d-old soil-grown plants and dissolved in RNase free water. Small sized RNAs were enriched by adding the same volume of 8M LiCl and centrifuging at 12,000rpm for 30 min at 4uC. RNA filter hybridizations were carried out as previously described [44]. LNA probe [45] was used for hybridization (59- cgagcAgtGgcG- gatCcaaga-39; uppercases represent modified nucleotides). Chromatin Immunoprecipitation (ChIP) Assays The ChIP assays were performed using 20-d-old soil-grown plants and as previously described [46]. Antibodies against H3K9me1 (07-450), H3K9me2 (07-441) and H3K9me3 (07-442) were from Upstate Biotechnology. Construction of RNAi Vector The genomic DNA from Col was used as a template for PCR amplification using the primer pairs (CX2004: ctcga- gATTTTTGTGGTAATATATATATA and CX2005: agatctA- CATCAATCCAAGTTCAAGC, carrying the XhoI and BglII sites, respectively). The PCR products were sequentially inserted into pUCC-RNAi vector using the XhoI/BglII and BamHI/SalI sites for both the sense and antisense orientations. The stem-loop structured fragment was cut off and further cloned into a modified pCambia1302 vector (pCambia1302-LX-1) and used for plant transformation (XF718). All transgenic plants used for further analyses had been tested for their successful de novo methylation at MPF. DNA Methylation Analysis: Southern Blot, Bisulfite Sequencing, and McrBC-PCR Genomic DNA was isolated from rosette leaves of 4-week-old, soil-grown plants. Southern blots was performed as previously described [22] using PCR products amplified from FLC promoter as the probe (Figure 1). Bisulfite sequencing experiments were performed as previously described [47]. Primers with one end in FLC-TE and the other in FLC were designed to specifically amplify the FLC-TE and exclude other TEs in the genome. Only the cytosines within TE were counted for methylation analysis of FLC- TE in Figure 3. McrBC-PCR experiments were performed as previously described [34,47], Equal amounts of McrBC-digested Small RNA-Directed Epigenetic Natural Variation and non-digested DNA were used for PCR amplification. Real- time McrBC-PCR was performed to quantitatively measure the methylation level. The primer information for these experiments could be found in Supporting Information (Text S1). Bioinformatics After discarding smaller (,23 nt) and redundant sequences, 247,318 unique small RNA sequences in Col and 25,981 unique small RNA sequences in Ler were used for further analysis. All these siRNAs were mapped to the Col genome by BLAST [48] and PERL scripts, and the numbers of perfect matches were counted per 100 bp. Next, regions contain more than 3 hits within 300 bp in Ler but no hits in 1.5 kb at the same region in Col (Figure 6) were filtered out and overlapping regions were artificially combined. Col derived small RNA dataset was downloaded from NCBI GEO (GSE5228), and Ler derived small RNA sequences from NCBI GenBank (DQ927324-DQ972825). The Arabidopsis genome (Col) information was provided by TIGR (release version 5). Gene positions were annotated according to TAIR’s SeqViewer data. Tandem gene duplication information was provided by TIGR (tandem_gene_duplicates. Arab_R5). Supporting Information Figure S1 Sequence Alignment of MPF Region in Col and Ler. Gray shades indicate the polymorphism; green box indicates the hAT element insertion; red region indicates the TSDs (Target Site duplication); blue region indicates the TIRs (Terminal Inverted Repeats). Found at: doi:10.1371/journal.pgen.1000056.s001 (10.02 MB DOC) Figure S2 Bisulfite Sequencing Analysis of DNA Methylation at the MPF in kyp-2 (A), dcl1-9 (B), and FLC-TE in Ler (C). The x axis represents the position of the cytosines within the sequencing region; n indicates the number of the sequenced clones. The B4 region spans the junction between TE (white box) and the first intron of FLC (gray box). Only the cytosines within TE were counted for methylation analysis of FLC-TE in Figure 3. Found at: doi:10.1371/journal.pgen.1000056.s002 (9.01 MB TIF) Figure S3 DNA Methylation Analysis of MPF among Arabidopsis Accessions using McrBC-PCR. (A) Summary of the TE insertions at the first intron of FLC in different ecotypes. The number under each accession represents the length of the TE insertion. (B) Accessions reported to contain transposable element inserted in the first intron of FLC. (C) Accessions that are closely related to Ler. Di-1 and La-0 do not contain the FLC-TE insertion. TE (methylated) and Actin (unmethylated) serve as controls for the McrBC-PCR assay. Found at: doi:10.1371/journal.pgen.1000056.s003 (7.89 MB TIF) Figure S4 Genomic Southern Blot Analysis for the Copy Number of hAT Element in Col and Ler. Genomic DNAs from both Col and Ler were digested by EcoR V, Hpa II and Nco I. A 160 bp region within the hAT element was PCR amplified and used as the probe for hybridization. Found at: doi:10.1371/journal.pgen.1000056.s004 (13.48 MB TIF) Figure S5 Target DNA Methylation to MPF in Col using RNAi Approach. (A) A diagram shows the 202 bp fragment used for the construction of the RNAi vector. (B) Flowering time analysis for the RNAi transgenic lines (T0 generation); each individual PLoS Genetics | www.plosgenetics.org 9 April 2008 | Volume 4 | Issue 4 | e1000056