Fig. 1

The workflow and terminology used to analyze and describe cold-dependent differential methylation in sugar beet. a Leaf material of two sugar beet accessions grown under control conditions or exposed to cold with three replicates per genotype and growth condition was collected. Sample material was split for extraction of RNA, or DNA, and subsequent RNA- (RNA-seq), or whole-genome bisulfite sequencing (WGBS), respectively. Raw reads were trimmed and the cleaned reads were mapped to the reference genome (Refbeet-1.2.2; [22]). For transcriptome analysis, mapped reads were quantified on gene level and the extracted data used to identify genes, differentially expressed (DEGs) between control and cold conditions. For methylome analysis, the number of methylated and unmethylated reads was extracted for each cytosine covered by at least 8 reads in each sample. From this data, methylation levels were calculated [methylated reads / (methylated + unmethylated reads)] and further analyzed in order to identify cold-dependent differential methylation affecting single cytosines (DMCs), or occurring along longer stretches of DNA (differentially methylated regions = DMRs) comprising at least three cytosines assigned to the same sequence context. To integrate transcriptomic and methylomic data, DEGs were functionally classified to identify cold-regulated, putative facilitators of DNA methylation, or DNA demethylation. Further, correlations between gene expression and methylation levels, or of changes in expression and changes of methylation were analyzed. b Schematic representation of context-specificity of DNA methyltransferases and demethylases. c Schematic representation of terms used to describe hypo (blue), or hyper (red) methylation, and differential methylation of individual positions (DMC) or differentially methylated regions (DMRs)