Population-level analysis reveals the widespread occurrence and phenotypic consequence of DNA methylation variation not tagged by genetic variation in maize.

BACKGROUND: DNA methylation can provide a source of heritable information that is sometimes entirely uncoupled from genetic variation. However, the extent of this uncoupling and the roles of DNA methylation in shaping diversity of both gene expression and phenotypes are hotly debated. Here, we investigate the genetic basis and biological functions of DNA methylation at a population scale in maize. RESULTS: We perform targeted DNA methylation profiling for a diverse panel of 263 maize inbred genotypes. All genotypes show similar levels of DNA methylation globally, highlighting the importance of DNA methylation in maize development. Nevertheless, we identify more than 16,000 differentially methylated regions (DMRs) that are distributed across the 10 maize chromosomes. Genome-wide association analysis with high-density genetic markers reveals that over 60% of the DMRs are not tagged by SNPs, suggesting the presence of unique information in DMRs. Strong associations between DMRs and the expression of many genes are identified in both the leaf and kernel tissues, pointing to the biological significance of methylation variation. Association analysis with 986 metabolic traits suggests that DNA methylation is associated with phenotypic variation of 156 traits. There are some traits that only show significant associations with DMRs and not with SNPs. CONCLUSIONS: These results suggest that DNA methylation can provide unique information to explain phenotypic variation in maize.

Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

Post-conversion targeted capture of modified cytosines in mammalian and plant genomes.

We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.

Genetic perturbation of the maize methylome.

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.

Consistent and heritable alterations of DNA methylation are induced by tissue culture in maize.

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.

Inheritance patterns and stability of DNA methylation variation in maize near-isogenic lines.

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance. DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.

Epigenetic and genetic influences on DNA methylation variation in maize populations.

DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

Heritable epigenetic variation among maize inbreds.

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation.

Nonadditive expression and parent-of-origin effects identified by microarray and allele-specific expression profiling of maize endosperm.

Plant endosperm cells have a nuclear ratio of two maternal genomes to one paternal genome. This 2 to 1 dosage relationship provides a unique system for studying the additivity of gene expression levels in reciprocal hybrids. A combination of microarray profiling and allele-specific expression analysis was performed using RNA isolated from endosperm tissues of maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal hybrids at two developmental stages, 13 and 19 d after pollination. The majority of genes exhibited additive expression in reciprocal hybrids based on microarray analyses. However, a substantial number of genes exhibited nonadditive expression patterns, including maternal like, paternal like, high parent like, low parent like, and expression patterns outside the range of the parental inbreds. The frequency of hybrid expression patterns outside of the parental range in maize endosperm tissue is much higher than that observed for vegetative tissues. For a set of 90 genes, allele-specific expression assays were employed to monitor allelic bias and regulatory variation. Eight of these genes exhibited evidence for maternally or paternally biased expression at multiple stages of endosperm development and are potential examples of differential imprinting. Our data indicate that parental effects on gene expression are much stronger in endosperm than in vegetative tissues.