transgenic line was confirmed for their de novo methylation at MPF. (C) FLC expression analysis by real-time RT-PCR using the seedlings of one T2 transgenic line (homozygote for the transgene) which had been confirmed for its methylation at MPF. Found at: doi:10.1371/journal.pgen.1000056.s005 (9.20 MB TIF) Figure S6 Cluster Analysis. Small RNA hits were counted per 100 bp of a 1.5 kb range in Ler at the 68 loci identified in this study that have no less than 3 unique ,24 nt siRNA matches within 300 bp (show in the central) and meanwhile no hits in a 1.5 kb region in Col (Figure 4). Found at: doi:10.1371/journal.pgen.1000056.s006 (7.40 MB TIF) Figure S7 Genome-wide Distribution of the 68 loci. Black bars represents loci with 3 to 5 hits within 300 bp; blue bars represents loci with 6 to 8 hits within 300 bp; red bars represents loci with more than 9 hits within 300 bp. Black rectangles represent the centromeric region. Found at: doi:10.1371/journal.pgen.1000056.s007 (10.03 MB TIF) Figure S8 RNA-directed DNA Methylation at Locus #10. (A) The siRNAs matched to this region. (B) Bisulfite sequencing results summarized in different sequence contexts; the x axis represents the position of the cytosines within the sequencing region; n indicates the number of the sequenced clones. The color coding of the cytosines in (A) matches the legend in (B). Found at: doi:10.1371/journal.pgen.1000056.s008 (4.26 MB TIF) Figure S9 DNA Methylation Analysis using McrBC-PCR. McrBC cuts at methylated sites in the template DNA, therefore resulting in attenuated PCR products for methylated loci; however, the PCR amplification of unmethylated loci will not be affected by McrBC digestion. (A) ‘‘Locus’’ represents the locus number tested from among the 68 loci that passed our filters; References 1. Goldberg AD, Allis CD, Bernstein E (2007) Epigenetics: a landscape takes shape. Cell 128: 635–638. 2. 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Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20: 3407–3425. Small RNA-Directed Epigenetic Natural Variation ‘‘hits’’ represents the unique siRNA hits within each 300 bp region. Locus #60 with the methylation signal in Col (Table S1) is also methylated in Ler. (B) The negative (Actin) and positive (MPF and FLC-TE) controls for McrBC-PCR. The 1.2 kb methylated FLC-TE is only present in Ler, therefore the PCR products (using primers matched to FLC on both sides of the TE but not within itself) from Ler derived samples are 1.2 kb larger than those from Col derived samples. Found at: doi:10.1371/journal.pgen.1000056.s009 (6.99 MB TIF) Table S1 The 68 Loci Identified in this Study. Found at: doi:10.1371/journal.pgen.1000056.s010 (0.12 DOC) MB Table S2 Basic Information of the Genes Corresponding or Adjacent to siRNA Clusters. 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Malagnac F, Bartee L, Bender J (2002) An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation. Embo J 21: 6842–6852. PLoS Genetics | www.plosgenetics.org 10 April 2008 | Volume 4 | Issue 4 | e1000056