Chilling requirement is a critically important phenological trait that controls the timing of vegetative bud break and floral bloom, and impacts the climatic distribution of temperate tree species. However, the molecular basis of the chilling requirement trait is not well understood. Analysis of chilling requirement QTL from peach and apricot has detected numerous genes known to modulate DNA and histone methylation pathways. Evidence from other plant systems suggests these types of epigenetic mechanisms may play a role in temperature-modulated plant developmental responses.
For this study, DNA from vegetative buds of overwintering peach trees was examined for changes in methylation status during chilling accumulation. Specifically, dynamic methylation changes between high chill (HC) and low chill (LC) genotypes and changes within genotypes were analyzed.
For this purpose, a methylation sensitive amplified fragment length polymorphism (MS-AFLP) technique was used to examine cytosine methylation status of vegetative bud DNA. Differences in anonymous CCGG regions of the genome were detected using isoschizomer restriction endonucleases (MspI and HpaII), which are differentially sensitive to methylation.
AFLP results showed multiple time points where methylation patterns differ between LC and HC vegetative bud samples. Re-amplified AFLP bands were cloned and sequenced to examine the nature of the sequences undergoing methylation changes during dormancy and locate them on the peach genome. From this analysis, four different sequences appeared to undergo differential changes in methylation. They are: 1) a portion of the coding sequence of a COBRA-like gene, 2) a portion of an unknown gene containing aVQ Motif, 3) a sequence ~ 200bp upstream from the start site (ATG) of a phytoceramidase gene and 4) a sequence 10 bp downstream from the stop codon (TAA) of a DNA repair helicase (DEAD_2) in the DUF 1227 super-family. Of these four sequences, one was in a chilling QTL on LG1 and other was in a bloom date QTL on LG1. The other two were adjacent to chilling QTLs on LG1 and LG8.