Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 197 more ABI19, potentially prolonging grain filling and increasing seed size (consistent with the allele effect). This candidate regulatory element now merits functional testing (e.g. reporter assays or genome editing to swap alleles). Another striking example involves the NKD1 locus and a trait not immediately obvious: leaf length. A GWAS had identified a SNP in the promoter of NKD1 that was associated with variation in maize leaf length (a vegetative trait). This SNP lies within an OCR (the NKD1 promoter is accessible during seed development as we discussed) (Zhu et al., 2024). One hypothesis is that the NKD1 allele that affects leaf length does so indirectly via seed development – possibly by altering aleurone function or hormone balance in the seed that has carry-over effects on seedling vigor and leaf growth. To test the effect of this regulatory SNP, we used CRISPR-Cas9 genome editing to create small deletions in the NKD1 promoter that encompassed the SNP site (essentially generating NKD1 promoter edits denoted NKD1pro-1 and NKD1pro-2 in our study). These edited lines had subtle but clear phenotypic differences: their third and second leaves (14 days after germination) were significantly longer than those of wild-type plants. This phenocopies the association of the particular SNP allele (longer leaves), thereby confirming that the regulatory region containing that SNP influences leaf development. Our ATAC-seq data showed that the edited region is normally an active promoter OCR in seeds; altering it likely changed NKD1 expression in the seed (though NKD1 is mostly seed-specific, so the mechanistic connection to leaves might be via altered seed nutrient profile or hormone provisioning). Nonetheless, this result demonstrates that manipulating an open chromatin region identified in seed development can have measurable trait outcomes, underscoring the value of open chromatin maps for pinpointing such elements. We also identified candidate regulatory elements for seed composition traits. For example, oil content in maize kernels is largely determined by the embryo (which is rich in lipids). A major QTL for oil content is located on chromosome 6 (the qHO6 locus). Our chromatin map revealed an enhancer region within that QTL interval that becomes accessible specifically in the embryo during mid-development. This enhancer is near a gene encoding an AP2-domain transcription factor that is expressed in the embryo and known to affect lipid accumulation (potentially an ortholog of WRI1, an AP2 factor controlling oil biosynthesis in Arabidopsis). The presence of an embryo-specific OCR at this locus suggests this could be the causal regulatory region for the oil QTL; SNPs altering this enhancer’s strength might lead to differences in expression of the AP2 factor and subsequently lipid biosynthesis genes. This hypothesis is reinforced by the motif content – the enhancer contained multiple RY motifs (which AP2/VRN1 factors might be part of a complex with ABI3), hinting at complex regulation. We consider this enhancer a top candidate for fine-mapping and editing to boost oil content (Zhou et al., 2024b). 6.3 Epigenetic accessibility as a guide for genome editing The knowledge we learn from open chromatin can truly assist in crop breeding, especially when using tools such as CRISPR. ATAC-seq can show which parts of DNA are active. These active sites are ideal locations for editing, which may change the way genes work or the traits of plants. If we do not want to change the gene itself, we can edit nearby open regions, such as promoters or enhancers. These regions control the frequency of gene usage. Our research results support the concept of "cis engineering". This means using CRISPR/Cas to adjust these control sites, thereby altering the behavior of genes without touching the genetic code. One of the clearest demonstrations in our study is the NKD1 promoter editing discussed above. By deleting a small region of an accessible promoter containing a trait-associated SNP, we were able to mimic the effect of a natural variant and confirm its phenotypic influence. Notably, the edited plants did not show obvious deleterious effects on seed development or plant viability, suggesting that fine-tuning regulatory regions can produce a subtle improvement in a trait (leaf growth in this case) without negative trade-offs. This is a promising outcome for breeding – it implies that targeting regulatory alleles (as opposed to complete knockouts of genes) might yield beneficial phenotypic variation that is less likely to harm overall fitness, because it’s making quantitative rather than qualitative changes in gene expression. Another scenario in which open chromatin data guide editing is when creating synthetic promoters or enhancers. If a certain pathway needs to be upregulated, one might want to insert an enhancer or strengthen an existing one.
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