Fig. 1

Hybrid-Induced DNA methylation changes are transgenerational through multiple generations of backcrossing and selfing. a Schemes for producing genetic materials. F₁ hybrid (BM) was made between B73 (maternal parent) and Mo17 (as pollen donor) and backcrossed consecutively with Mo17 (recurrent parent) to produce backcross lines from BM1 (F₁BC1) to BM5 (F₁BC5); BM6 (F₁BC6) was subsequently self-pollinated as BM6S1 for three generations to produce BM6S3. Likewise, the reciprocal F₁ hybrid MB underwent a similar backcrossing scheme with B73 pollen to produced MB1 (F₁BC1), MB3 (F₁BC3), MB5 (F₁BC5), and MB6 (F₁BC6) that was self-pollinated to produce MB6S1-MB6S3. Large circles indicate epigenome (methylome) of B73 (light blue) and Mo17 (yellow), and small circles indicate nuclear genomes of B73 (blue) and Mo17 (orange). b Kernel density distribution of relative methylation changes (y-axis) in the F₁ hybrids compared to the mid-parent value (MPV) and in the backcross lines compared to their recurrent parents. The density is estimated by the difference (I-II) divided by II (with a 100-kb bin window). Line colors indicate BM (black), MB (gray), BM3 (light yellow), BM5 (yellow), BM6S1 (orange), and BM6S3 (red). Double asterisks indicate statistical significance (P < 0.01, two-sided Wilcoxon signed-rank test). c Heatmaps show CHH methylation changes in the TCM and TCdM loci (left) and two examples (right). Methylation levels were increased in 6757 TCM loci (upper panel) and decreased in 4307 TCdM loci (lower panel) in all backcross and selfing lines examined. Examples are Chr1: 167,654,401-167,654,600 (TCM, upper panel) and Chr5: 118,251,401-118,251,600 (TCdM, lower panel). Numbers in parentheses indicate average methylation levels for the DMRs. Double asterisks indicate statistically significant changes between a F₁ hybrid and MPV or between a backcross line and its recurrent parent (P < 0.05, one-way ANOVA test)