The study of accessible chromatin, also known as open chromatin, is currently a hot spot in the research of chromatin non-coding cis-regulatory elements and cis-trans controls of gene expression. Compared to animals, the accessible chromatin is different and relatively conserved across plant species. The identification of accessible chromatin regions (ACRs) in plants promotes our understanding of gene regulation, plant development, and regulatory changes underlying phenotypic evolution. Here, we describe an approach to identify wheat ACRs using differential MNase-seq. Micrococcal nuclease (MNase) is highly sensitive to digestion degree; it tends to cut accessible regions in case of light digestion and more closed regions in case of heavy digestion. We set up gradients of high- and low-concentration MNase digestion and performed high-throughput sequencing of DNA fragments near the length of mononucleosomes in the fragments digested by the two gradients. By comparing the differences in read enrichment under the two concentrations, we defined wheat genome regions highly sensitive to the change of digestion degree as ACRs and regions highly insensitive to the change as closed chromatin regions and identified nucleosome occupancy profiles as well. In short, we modified and refined the method from Rodgers-Melnick et al. (2016) for identifying open chromatin in maize, optimizing the nuclei extraction and ACRs identification for polyploidy, making its application in plants more intuitive, fast, and easy to operate. This method allows us to use MNase-seq to more easily identify ACRs in polyploid plants or large-genome species and to make multiple comparisons with ACRs obtained by other methods, so as to better facilitate the study of plant ACRs.

Graphical overview