24-nt-phasiRNA associated CHG/CHH DNA methylation contributes to growth heterosis of Populus deltoides

24-nt siRNA-directed DNA methylation is crucial for heterosis in herbaceous plants, but its regulatory function, particularly 24-nt phasiRNA (phased small RNA)-mediated DNA methylation, remains unexplored. This study analyzed whole-genome DNA methylation and small RNA expression between high-growth (H2) and low-growth (L3) Populus deltoides hybrids and their parents, investigating how 24-nt phasiRNA-associated DNA methylation contributes to heterosis. Methylation levels in hybrids and parents ranged from 11.30% to 12.30%, with 22.27% to 23.34% of methylation sites being hybrid-specific. Differentially methylated regions (DMRs) between hybrids and parents mainly originated from similarly methylated regions in parents. Hypo-DMRs were more prevalent in H2, which was the opposite in L3. In the promoter region, expression of differentially methylated genes was correlated with DMR methylation levels between hybrids and parents, albeit exhibiting opposite trends in H2 and L3. Approximately 9% of 24-nt phasiRNAs in hybrids showed additive, dominant, or over-dominant inheritance, with H2 showing paternal bias and L3 maternal bias. 24-nt phasiRNAs were positively correlated with promoter CHG/CHH-DMR methylation levels and negatively correlated with non-additive gene expression, which may facilitate heterosis formation. Gene expression associated with 24-nt phasiRNAs-linked DNA methylation in hybrids was distinctly different, with H2 mainly showing high 24-nt phasiRNA expression, hyper-DMRs, and decreased gene expression, while L3 mainly showed low 24-nt phasiRNA expression, hypo-DMRs, and increased gene expression. We identified 13 candidate 24-nt phasiRNAs and seven key genes, specifically involved in nitrogen response (NLP2/NLP8-like) and photosynthesis (PsbP), which may play an important role in heterosis. This study provides the first evidence of 24-nt phasiRNA-associated DNA methylation in tree heterosis, offering novel insights into epigenetic modifications.