Maize Genomics and Genetics 2025, Vol.16, No.6, 325-333 http://cropscipublisher.com/index.php/mgg 331 of salt-tolerant materials is more "flexible", while the changes of some sensitive strains seem rigid and unable to keep up with the environmental rhythm. Some key genes, such as ZmKTF1, have a slight "problem" - a mutation, the methylation level of CHH drops immediately, and as a result, the salt sensitivity also increases (Wang et al., 2024). So, some loci are not just for show; they are indeed the main forces involved in regulation. In the broader population analysis, researchers have identified more than one hundred suspected candidate regions by integrating isolated population and transcriptome data, among which there are no shortage of genes closely related to membrane function, ion channels or ROS clearance mechanisms (Maimaiti et al., 2025). 6.3 Case study on methylation-expression correlation of candidate genes Just looking at sequencing data often fails to reveal the essence - differences do not necessarily mean they are effective. What truly explains the issue lies in observing the "response actions" of genes under stress. Take Zm00001d053925 as an example. This gene is strongly induced when exposed to salt stress in the salt-tolerant strain AS5. However, when it is replaced with NX420, its expression is very calm and there is almost no response (Zhu et al., 2023). If you take another look at its methylation data, you will find that the hypomethylated region at the root is precisely located upstream of it. This relationship of "less methylation and higher expression" is no coincidence. Similar are some oxidoreductase genes regulated by ZmKTF1, which can help regulate ROS levels and relieve cellular stress. Behind these functions, there are also significant methylation differences. Not all genes cooperate in this way, and not every case can be summarized into a pattern. However, these phenomena do reflect a notable point: Sometimes, whether a gene can "speak out" in adverse circumstances may be driven by epigenetic modifications. 7 Application Prospects of DNA Methylation Research in Salt-Tolerance Breeding of Maize DNA methylation has been mentioned by many people in recent years, especially in the field of salt-tolerant breeding of corn, where discussions have been held on whether it is a new type of selection tool worth investing in. However, for it to be truly useful, it not only needs to be reliable on its own but also depends on whether it can be well integrated with conventional methods such as GWAS and QTL mapping. Candidate genes related to salt tolerance, such as ZmCLCg and ZmPMP3, have long been identified through localization and have been used as targets for MAS in many studies. If methylation data can be linked with these SNP or QTL information, the epigenetic levels that traditional genetic markers cannot capture may be "filled", especially in complex traits like salt tolerance that are influenced by both the environment and genes. Of course, not all methylation changes are guaranteed by "genetic transmission", and not every variety will show significant methylation differences under environmental stress. This point must be recognized. However, this does not prevent us from looking for those relatively "stable" methylation patterns at some key regulatory sites. Once found, it will be a breakthrough for the rapid screening of salt-tolerant materials. In fact, current research has identified many regions and genes that may be related to salt tolerance through population segregation analysis and methylomics sequencing. Factors like ZmKTF1 have also been found to regulate the stress response through the RNA-mediated DNA methylation pathway, which further enhances the role of epigenetics in salt-tolerant breeding. If we take it a step further and introduce tools like CRISPR/dCas9 to perform targeted "operations" on certain methylation sites, future breeding methods may be more flexible and precise than currently imagined.. Ultimately, to truly make good use of epigenetic information, relying solely on a single omics is far from sufficient. Genomic, transcriptomic, methylomic and even metabolomic data must be interconnected in order to piece together the complete regulatory network behind salt tolerance. By means of this multi-omics integration approach, we can more confidently screen out key markers that have both genetic basis and epigenetic features, and further optimize the combination of alleles. In the future, breeding platforms are likely to combine high-throughput methylomics sequencing with SNP genotyping and transcriptome expression, selecting the most stable and adaptable materials from the very beginning to accelerate the breeding of corn varieties that can truly resist salt and withstand future climate change. Acknowledgments We are grateful to Dr. W. Wu for his assistance with the serious reading and helpful discussions during the course of this work.
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