Figure 5
Repression of A. arenosa MIR172b and its neighboring genes in Arabidopsis allotetraploids. (a) Diagram of an ∼55-kb genomic region containing MIR172b and 13 neighboring genes. Arrows indicate the orientation of gene transcription in each locus. Plus (+), minus (−) and ‘S’ indicate expression, low expression and silencing or undetectable in RT-PCR and SSCP assays. The expression states suggest putative left (L) and right (R) borders of the repression domain surrounding A. arenosa-derived MIR172b. The domain containing silenced A. arenosa loci in two allotetraploids is boxed. (b) Repression of A. arenosa MIR172b is associated with DNA methylation in the promoter regions. The genomic DNA located at ∼400-bp upstream of the transcription start site (Xie et al., 2005) was treated with bisulfite and amplified by PCR. The PCR products were purified and cloned, and individually cloned fragments were sequenced. The percentage of CG and CHG (H=A, T or C) methylation is calculated by the number of fragments containing methylated cytosines divided by the total number of fragments analyzed, as shown by the numbers near the promoters (Supplementary Table S5). Open and filled lollipops indicate unmethylated and methylated cytosines, respectively. Open, filled and slashed boxes indicate promoter regions, MIR172b sequences and coding regions of the two neighboring loci, respectively. Arrows indicate transcriptional orientation. AlloF1-At, AlloF1-Aa, As-At and As-Aa indicate A. thaliana (At) and A. arenosa (Aa) alleles in F1 resynthesized allotetraploids (AlloF1) and natural A. suecica (As), respectively.
