Figure 5

Schematic illustration of the promoter region and the methylation frequencies within the 35S promoter. A, Within the transfer DNA (T-DNA) the resistance marker (hptII) is driven by a nopaline synthase (NOS) promoter and confers resistance to the antibiotic hygromycin B. The gene of interest (GOI) is driven by a cauliflower mosaic virus promoter (35S). For methylation analysis a 294 bp fragment of the NOS promoter or a 346 bp fragment of the 35S promoter were amplified with the indicated bisulfite primers. Positions of the restriction sites of XbaI and EcoRV are indicated. B, Graphical output of the 35S promoter wide methylation in two consecutive generations of line PNA 1.2 (T₂ and T₃ stage). DNA was isolated from seedlings 15 days post germination (dpg). Filled symbols indicate cytosine methylation, whereas empty symbols indicate a lack of methylation. Red circles represent CG sites, blue squares represent CHG sites and green triangles represent CHH sites, whereas H = A,T or C. Analysis was performed with CyMATE [127]. The upper part illustrates the spatial distribution of the methylation sites within the promoter sequence. Mean cytosine methylation was calculated for the three methylation sites (CG, CHG and CHH) from individually picked clones (±SEM, n = 12 clones).