Reduced Representation Bisulfite Sequencing in Maize.

DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution ( Cokus et al., 2008 ). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies ( Smith et al., 2009 ). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants ( Hsu et al., 2017 ). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.