Methylome differences among barley inbreds and their association with genomic, transcriptomic, and phenotypic variation.

Epigenetic variation can play a crucial role in explaining the missing heritability of complex traits. To investigate genome-wide methylation in spring barley (Hordeum vulgare subsp. vulgare), we performed whole genome bisulfite sequencing on 23 parental inbreds from a community resource for genetic mapping. Our objectives were to characterize methylation variation, explore its association with single nucleotide polymorphisms (SNPs), and examine links to gene expression. The barley genome showed high average methylation levels of 88.6%, 58.1%, and 1.4% in the CpG, CHG, and CHH contexts, respectively. We identified nearly 500 000 differentially methylated regions (DMRs), with 64%, 64%, and 83% of DMRs in CpG, CHG, and CHH contexts, respectively, not associated with sequence variation. Around 6% of all DMRs showed significant associations with gene expression, with the direction of the correlations varying based on the DMR's location relative to the gene with a recognizable pattern. We exemplified this association between DNA methylation and gene expression on the known flowering promoting gene VRN-H1, identifying a highly methylated epiallele linked to earlier flowering. Lastly, methylation improved the prediction abilities of genomic prediction models for various traits, outperforming models based solely on SNPs and gene expression. These findings emphasize the independent role of DNA methylation to sequence variation.