DNA methylation consists of 5-methylcytosine and N6-methyl deoxyadenosine (6mA) and is crucial in plant development. However, its specific role and potential mechanism to initiate cotton fibers remain unclear. This study employed Oxford Nanopore Technologies (ONT) sequencing to analyze DNA methylation alterations in ZM24 and ZM24 fuzzless-lintless (ZM24fl) during fiber initiation. Our results indicated that DNA 6mA methylation exhibited the most remarkable difference among ovule samples at -2, 0, and 5 d post anthesis of ZM24 and ZM24fl. Subsequently, genes with significant changes in DNA 6mA methylation and transcription during fiber initiation were screened. We found that GhMAF1 displayed significant transcriptional upregulation and 6mA enrichment in its promoter, which could serve as a potential target for DNA 6mA in fiber initiation. Further, we knocked out GhMAF1 using CRISPR-Cas technology and demonstrated that GhMAF1 specifically promotes the initiation of fiber cells at the base of the ovule by mediating the downstream JAZ2/CPC-MML3/MML4 pathway. These findings unveil a novel spatial module of fiber cell initiation on the ovule surface that involves GhMAF1. Ultimately, this work provides significant knowledge for the regulatory network of DNA 6mA modification in fiber initiation to improve fiber yield and quality.
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