Fig. 4. Inbreeding-induced hypermethylation is reversed by random mating and analysis of ZmTCP binding affinity to methylated TBS.

(A) K-means clustering of CHH methylation levels in hyper-DMRs among S7, S11, high (H), and low (L) vigor M11 lines. (B) K-means clustering of expression levels of the genes associated with reversed DMRs. (C) GO-enriched terms of the genes with reversed expression levels (P < 0.05 and counts of ≥5). (D) Reduced binding affinity of recombinant ZmPCF1 protein to methylated probes containing TBS motifs (bold letters) in EMSA. Competition assays used unmethylated probes (competitor^Um, 100× and 500× excess), methylated probes (competitor^Me, 100× and 500× excess), unmethylated mutant probes (mutant-probe^Um, 500× excess), or methylated mutant-probes (mutant-probe^Me, 500× excess). CHH methylation sites (lollipop) are shown in the competitor^Me sequence. The band shift is indicated by the arrow. (E) DNA affinity purification (DAP)–qPCR analysis of recombinant ZmTCP proteins using genomic DNA of S7 and S11 (gS7 and gS11) and PCR-amplified genomic DNA from S7 and S11 (ampS7 and ampS11). Diagram of the Zm00001d015449 promoter region (3-kb upstream) shows locations of two TBS motifs (red arrowheads), CHH hyper-DMR (gray box), and the fragment (short black line) used in DAP-qPCR assays. Numbers shown are relative to the TSS (+1). Ratio of GST-ZmTCP/GST ≥ 5 was used to show binding of ZmTCP protein to the TBS fragment using the purified GST protein as the negative control. Different letters above each column indicate statistical significance at P < 0.05 (Tukey’s test).