Estimation of DNA Methylation in Wheat Using Reverse Phase Ultra-High-Performance Liquid Chromatography Coupled with Mass Spectrometry During Leaf Rust Pathogenesis.

Reverse phase ultra-high-performance liquid chromatography is considered the most reliable and sensitive technique to determine and separate analytes with small differences in their molecular weights. Leveraging this technique coupled with mass spectrometry, we developed a method for simultaneous determination of 2'-deoxycytidine and its methylated analogue, 5-methyl-2'-deoxycytidine, in wheat. The nonpolar C18 column was selected that greatly shortened the detection time to approximately 2 min. Formic acid was added to the mobile phase to achieve the complete separation. Thus, avoiding the signal suppression and interference of 2'-deoxycytidine in the detection of 5-methyl-2'-deoxycytidine. The method utilized a least toxic solvent system consisting of water and methanol. Although, DNA methylation can be detected in numerous ways, bisulfite sequencing achieve single-base resolution and methylation-sensitive amplified polymorphism is effective in pattern analysis. Whole-genome and reduced representation bisulfite sequencing provide more detailed or more targeted methylation profiles, respectively. However, these methods are data and cost-intensive and provide incomplete information due to DNA degradation. Methylated DNA immunoprecipitation sequencing is biased toward highly methylated regions, and ELISA is more high-throughput but less precise. Therefore, the optimized method, is specific, reliable, and highly sensitive for the detection and quantification of cytidine methylation levels in wheat during leaf rust pathogenesis.