Small RNA-Directed Epigenetic Natural Variation Figure 5. DNA Methylation Analysis in the F1 Heterozygous Plants from the Reciprocal Crosses between Col and Ler. (A) Bisulfite sequencing analysis at MPF (B1 region, see Figure 2) of four heterozygous lines from both the crosses of ColR6Ler= and LerR6Col=. SNPs at MPF between Col and Ler (see Figure S1) were used to distinguish the Col- and Ler-derived sequences from the heterozygous plants. ‘‘n’’ indicates the number of sequenced clones. The DNA methylation status was further confirmed by real-time McrBC-PCR using the McrBC non-digested (white bar) and digested (black bar) DNA from the heterozygotes (same to the DNA samples used in bisulfite sequencing). (B) Real-time McrBC-PCR analysis in 24 more lines from each direction of the crosses to test their methylation status at MPF. doi:10.1371/journal.pgen.1000056.g005 De novo Methylation at MPF Does Not Alter the Flowering Behavior of Col The 1.2 kb FLC-TE, when inserted into a Col FLC genomic construct, is sufficient to cause reduced expression of FLC in the transgenic lines [19], therefore, it is unclear whether the MPF has any functional relevance in FLC expression. Interestingly, FLC-Ler could strongly suppress the late flowering phenotype induced by FRIGIDA (FRI) and luminidependens (ld), but remains moderately sensitive to other mutants that up-regulate FLC like fca, fve, and fpa [37]. Recently, SUPPRESSOR OF FRI4 (SUF4) has been shown to bind to the promoter of FLC and directly interact with FRI and LD [38]. Moreover, FLC-Ler is again sensitive to FRI in a hen1-1 background [22] suggesting reversible epigenetic alteration might account for this weak response. To address the role of the epigenetic variation at MPF in flowering time control, we used an RNAi approach to artificially methylate MPF in Col, the ecotype in which MPF is originally unmethylated. All transgenic plants used for further analyses had been tested for their successful de novo methylation at MPF by McrBC PCR (data not shown). Both flowering time and FLC expression analysis showed that de novo methylation at MPF does not alter the flowering behavior of wild type Col (Figure S5). However, since Col is an early flowering ecotype and its FLC expression level is relative low, we can not rule not the possibility that MPF may play a more prominent role in some late flowering backgrounds with higher FLC levels, like FRI or ld. Genome-Wide Identification of ,24 nt siRNAs Directed Epigenetic Natural Variation The identification of MPF-siRNAs in Ler- but not Col-derived small RNA data made us wonder whether other loci are differentially and specifically matched by ,24 nt siRNAs in these ecotypes. Because the MPSS small RNA sequencing data are not readily comparable with the 454 data (due to length differences in the sequencing reads), the small RNA datasets we used for a genome-wide identification are all 454 sequencing data, derived from two recent studies: 247,318 unique small RNA sequences from Col [16]and 25,981 unique small RNA sequences from Ler [15]. Also, to balance the enrichment of longer siRNAs in the sequencing results of AGO4 precipitated pool from Ler [15], we only selected for further analyses the siRNA reads of length no less than 23 nt, hence most of the miRNAs and short sRNAs are discarded from both the Col and Ler datasets. Since only the Col genome sequence is complete and the number of sequenced Col derived siRNAs is much greater than that of Ler, in this study, we only analyzed the regions matched by clusters of siRNAs present specifically in Ler, to exclude the interference of genetic alteration and also for higher reliability (please see materials and methods for details about the bioinformatic analysis). The unique siRNA sequences over 23 nt from both Col and Ler were mapped to the genome, respectively, and hits were counted in windows of 100 bp. Although the majority of the ,24 nt small RNA clusters are conserved between Col and Ler (data not shown), after combining the overlapping regions, 68 unique loci were identified (including the MPF, locus #57; Table S1). These all shared the characteristic that they were matched by at least three distinct siRNAs within 300 bp in Ler but there were no hits in 1500 bp around the same region in Col (see Figure 6 for an example). Most of these loci are MPF-like, in that the siRNA matches are restricted to a small region (Figure S6), and their distribution in the genome is quite dispersed (Figure S7). Twenty-two loci are within known genes, and the other 46 are in intergenic regions (Table S2). An search of methylation data in Col (http://signal.salk.edu/cgi-bin/methylome) [25] demonstrated that all of these loci except locus #60 (located in a highly methylated region longer than several hundred kb, Table S1) were clearly lacking methylation; in addition, 28 loci contain repeat-associated sequences with one end beginning close to or within the small RNA matching region, and 15 loci had matching MPSS small RNA tags [12] (Table S1). We had also searched the website of DNA methylation information on the fourth chromosome in both Ler and Col background (http://chromatin.cshl.edu/cgi-bin/ gbrowse/epivariation/) [2]. For the 13 loci (#44,56) we identified on the fourth chromosome, six loci are found with methylation signals in their data: five loci (#46, 49, 52, 54, 55) are found specifically methylated in Ler as expected; one locus (#53) is methylated in both ecotypes but with a much higher methylation signal in Ler comparing to Col. Overall, our results are well supported by the two independent studies on epigenomics and epigenetic natural variation [2,25]. We investigated the methylation pattern of locus #10 as an example using bisulfite sequencing. Extensive methylation was found in Ler (Figure S8), whereas the same region in Col remained unmethylated (data not shown). Other eight randomly selected loci were tested using methylation sensitive McrBC-PCR, and all of them, even those with the minimal number of three unique siRNAs, were methylated in Ler but not Col (Figure S9). Furthermore, we tested the methylation status of 44 loci (in which 42 have successful amplification results), including all the loci on Chromosome I and II,, by real-time McrBC-PCR (Figure 7A). From these analyses, 88% of the loci (37 out of 42) were found to be specifically methylated in Ler but not Col, and no locus was found only methylated in Col, strongly supporting the role of ,24 nt siRNA in triggering epigenetic natural variation (Figure 7B). For the features of these 68 loci showing evidence of small RNA-directed variation in DNA methylation, we looked at the genes either corresponding to or adjacent to these loci within less than 1 kb distance of flanking sequence. Among the 64 genes identified (some intergenic loci did not have flanking genes within 1 kb upstream and downstream), 22 genes were found matched by genic siRNA clusters; 18 genes contained siRNA clusters in their 59 region and 24 genes with clusters in 39 regions (Table S2). Among the 22 genic regions, six were transposable elements, consistent with the role of transposable element in epigenetic regulation [39]. Moreover, many of these genes are reported or predicted to have important functions (Table S2). Therefore, additional investigation of these genes may help us to understand the role of epigenetic alteration in evolution and natural variation. Discussion Natural variation is a fundamental aspect of biology, and the implications of natural variation for deciphering the genetics of PLoS Genetics | www.plosgenetics.org 6 April 2008 | Volume 4 | Issue 4 | e1000056