Transcriptome and methylome interactions in rice hybrids

Abstract

DNA methylation is a heritable epigenetic mark that controls gene expression, is responsive to environmental stresses, and, in plants, may also play a role in heterosis. To determine the degree to which DNA methylation is inherited in rice, and how it both influences and is affected by transcription, we performed genome-wide measurements of these patterns through an integrative analysis of bisulfite-sequencing, RNA-sequencing, and siRNA-sequencing data in two inbred parents of the Nipponbare (NPB) and indica (93-11) varieties of rice and their hybrid offspring. We show that SNPs occur at a rate of about 1/253 bp between the two parents and that these are faithfully transmitted into the hybrids. We use the presence of these SNPs to reconstruct the two chromosomes in the hybrids according to their parental origin. We found that, unlike genetic inheritance, epigenetic heritability is quite variable. Cytosines were found to be differentially methylated (epimutated) at a rate of 7.48% (1/15 cytosines) between the NPB and 93-11 parental strains. We also observed that 0.79% of cytosines were epimutated between the parent and corresponding hybrid chromosome. We found that these epimutations are often clustered on the chromosomes, with clusters representing 20% of all epimutations between parental ecotypes, and 2-5% in F1 plants. Epimutation clusters are also strongly associated with regions where the production of siRNA differs between parents. Finally, we identified genes with both allele-specific expression patterns that were strongly inherited as well as those differentially expressed between hybrids and the corresponding parental chromosome. We conclude that much of the misinheritance of expression levels is likely caused by epimutations and trans effects.

Fig. 1.

SNPs between NPB and 93–11. (A) Identification of SNPs required a minimum of three reads per strand in each parental strain. Although there is ambiguity because of cytosine conversion in bisulfite-treated DNA, we can resolve this by considering the sequence of the reverse strand, and, as a result, are able to call all possible types of SNPs. In the example, X indicates the location of the SNP and reads represent, in order, NPB forward strand, NPB reverse strand, 93–11 forward strand, and 93–11 reverse strand. (B) SNP distribution over the genome is plotted on the bottom in windows of 10,000 bases. Each color change represents a new chromosome. The top line represents the density of genes. (C) The fraction of the various types of SNPs found in NPB versus 93–11 is shown with the CT and GA SNPs grouped together, and they represent nearly 74% of all SNPs. (D) The relative frequency of each SNP between NPB and 93–11 is shown per 1,000 bases. Methyl-Cs convert to Ts at a rate that is nearly five times that of unmethylated Cs.

Fig. 2.

Epimutations. (A) NPB has slightly higher methylation levels at CG and CHG sites, whereas 93–11 has higher methylation levels at CHH sites. The methylation level of individual hybrid chromosomes is relatively unchanged compared to their parents. (B) The percentage of differentially methylated sites in each comparison in C (black), CG (red), CHG (green), and CHH (blue) contexts. The dark-red fraction of each bar indicates the percentage of epimutations that were also divergent between the parents. For the 93–11 versus NPB comparison, the red portion indicates the percentage that was overlapping in any of the transgenerational comparisons. (C) A typical epimutation cluster showing increased methylation in 93–11 and nearly absent methylation in NPB. There is also a region of differential methylation in the 93–11 chromosome of a hybrid and the 93–11 parent that cannot be attributed to a lack of coverage. 9 × N and N × 9 represent the reciprocal crosses between NPB and 93–11, and |N and |9 represent the NPB and 93–11 chromosomes within the hybrids. (D) Among epimutation clusters, as the fraction of methylation sites that are greater in NPB increases, so does the fraction of siRNAs that are found in NPB versus 93–11.

Fig. 3.

Transcript level changes. (A) Venn diagram showing the number of genes differentially methylated or differentially expressed between NPB and 93–11. (B) A metaplot of SNP density is displayed. Each gene was divided into 100 bins and the average SNP density is plotted in 100 bins upstream of the gene to 100 bins downstream of the gene. In allele-specific genes, SNP density increases over the genic region, whereas for all genes, it decreases. (C) Genes are similarly broken into bins as in B, and the epimutation density is plotted over all genes and allele-specific genes. As in the case of SNP density, the epimutation density increases over allele-specific genes compared to all genes.