The role of subgenome expression dominance and DNA methylation in the heterodichogamy of Juglans mandshurica.

Heterodichogamy enhances species diversity and adaptability by reducing inbreeding depression and sexual interference in plants. However, its epigenetic molecular basis remains underexplored. We identified 1877 potential heterodichogamy-related genes (HRGs) in Juglans mandshurica Maxim. and examined their associations with plant hormones, subgenome dominance, DNA methylation, and transposable elements (TEs) using RNA sequencing, whole-genome bisulfite sequencing (WGBS), and liquid chromatography tandem-mass spectrometry (LC-MS/MS). Our study highlights the impact of subgenome dominance and epigenetic modifications on HRGs expression patterns. We found that HRGs are closely linked to stress and hormonal responses. Notably, Trehalose-6-phosphate (T6P) metabolism-related genes are significantly enriched among HRGs, biased expression genes (BEGs), and heterodichogamy-related differentially methylated genes (HRDMGs). We also observed expression bias toward the dominant subgenome in flowers, with male flowers exhibiting more BEGs and thus higher stress resistance than female flowers. DNA methylation levels were higher in early flowering groups, suggesting a relationship between DNA methylation and early flowering. Additionally, DNA methylation of TEs, particularly long terminal repeat (LTR), terminal inverted repeat (TIR), and non-TIR types, may regulate the biased expression of neighboring HRGs, thereby influencing heterodichogamy. Our study reveals the epigenetic basis of heterodichogamy and the role of subgenome dominance in the reproductive adaptability of J. mandshurica.