Detection of differential DNA methylation in date palm (Phoenix dactylifera L.) vitroplants using whole genome bisulfite sequencing analysis

Date palm (Phoenix dactylifera L.) is an essential fruit crop in arid and semi-arid regions, where tissue culture is widely used for large-scale clonal propagation. However, this technique often generates somaclonal variants that deviate from original cultivar traits, largely due to epigenetic modifications such as DNA methylation. Here, whole genome bisulfite sequencing (WGBS) was applied to two widely cultivated Qatari cultivars, Khalas and Kheneizi, to compare cytosine methylation profiles between tissue culture-derived plants and offshoot-derived controls. Thousands of differentially methylated regions (DMRs) were identified. Khalas exhibited a predominance of hypomethylated regions, whereas Kheneizi showed slightly more hypermethylated regions. Across both cultivars, CG sites tended to lose methylation, while CHG sites more frequently gained methylation in tissue culture-derived plants. Genes overlapping with DMRs (DMGs) displayed cultivar-specific enrichment patterns. In Khalas, hypermethylated DMGs were mainly linked to nuclear organization, chromatin remodeling, and stress signaling, while hypomethylated genes were associated with defense responses and intercellular communication. In Kheneizi, hypermethylated DMGs were enriched in RNA metabolism, translation, and hormone signaling, whereas hypomethylated DMGs were associated with metabolism, phosphorylation-mediated regulation, and stress responses. KEGG enrichment further revealed shared involvement of RNA degradation, mRNA surveillance, aminoacyl-tRNA biosynthesis, and MAPK signaling pathways. These results demonstrate that tissue culture induces DNA methylation changes in date palm, potentially affecting development and contributing to off-type phenotypes. Understanding these epigenetic alterations provides valuable insights for improving clonal fidelity and optimizing tissue culture propagation in perennial crops.