The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.Maize exhibits a wealth of epigenetic phenomena, from transposon silencing, cycling, and presetting, to gene imprinting and paramutation. Furthermore, despite the complexity and sophistication of maize breeding, there is a large degree of “hidden” variation for many traits that is difficult to explain by allelic variation alone (Gottlieb et al. 2002). At least some of this unexplained variation might be due to epigenetic rather than genetic changes in the maize genome (Richards 2011). A recent study using anti-methylcytosine antibodies and microarray hybridization to detect DNA methylation demonstrated clear differences between maize inbred lines, lending support to this hypothesis (Eichten et al. 2011). Similarly, studies using genome-wide sequencing of methylation-dependent restriction fragments have revealed that most methylation is found in transposable elements, prime sources of such variation (Palmer et al. 2003; Wang et al. 2009). However, neither method had the capability to detect individual cytosines in their sequence context.In plants, cytosine methylation occurs in symmetric (CG and CHG, where H is A, C, or T) as well as asymmetric (CHH) contexts. Methylation in each context is associated with DNA replication, histone modification, and RNA interference, respectively, although these mechanisms overlap (Law and Jacobsen 2010). The maize genome comprises roughly 50,000 genes and more than 1 million transposons and related repeats (Schnable et al. 2009). Approximately 29% of the cytosine residues are methylated as 5-methylcytosine (Montero et al. 1992), mostly in transposons (Palmer et al. 2003). We have used very-high-coverage whole-genome bisulfite sequencing to explore DNA methylation at nucleotide resolution in genes, transposons, and other features of the maize genome, as well as its heritability and potential contribution to traits. We have found that methylation in different sequence contexts is guided differentially by small RNA and is correlated with transposon insertion and mRNA splicing. Heritable and predictable switches in DNA methylation were detected in recombinant inbred lines. These shifts were apparently triggered by small RNA, resembling paramutation, but then maintained by replication-dependent symmetric methylation.We present a high-resolution and high-coverage map comprising the methylation status of individual cytosines throughout the inbred maize genome. Using this resource, we demonstrate that methylome sequencing of recombinant inbred lines at much lower coverage is sufficient to detect widespread paramutation in the maize genome. Future studies using low-coverage methylome sequencing can take advantage of this resource to determine the impact of differentially methylated regions on gene expression, chromosome biology, and transgenerational inheritance. For example, this will allow breeders to determine the contribution of cytosine methylation to phenotypic variation among elite inbreds and hybrids, artificially induced chromosomal variants (such as doubled haploids), and clonally micropropagated strains, which are subject to such epigenetic variation (Richards 2011). Thus, breeders could deploy a form of “epigenomic selection,” analogous to genomic selection, by which individuals with desired epigenomic patterns could be retained in breeding programs.