DEVELOPING A NOVEL HYPOMETHYLATED POPULATION AND CHARACTERIZING A STABLE EARLY FLOWERING EPIMUTANT IN STRAWBERRY (FRAGARIA VESCA)

Abstract

DNA methylation, as one of the epigenetic marks, can be passed from generation to generation. Epigenetic regulation of phenotypic traits can be demonstrated by altering methylation patterns with DNA demethylating reagent 5-azacytidine (5-azaC). Woodland strawberry (Fragaria vesca, 2n=2x=14) has a rapid generation time (3.5 months in the greenhouse), small plant stature and small sequenced genome size (~240 MB). F. vesca makes an ideal model system for genomics analysis and gene functional study in the Rosaceae family. In order to obtain a F. vesca population with the same genetic background but increased variation for epigenetic information, 5-azaC was used to generate a hypomethylated population by treating highly inbred seeds from Hawaii 4 (H4S8). The uniform genetic background was first confirmed using Amplified Fragment Length Polymorphism (AFLP). Additionally, whole genome sequencing demonstrated the epimutagenic, not mutagenic, effect of 5-azaC resulting in primary DNA sequence changes. Subsequent detailed phenotypic assessments measured flowering time, rosette diameter and stolon emergence time. Expanded variation in these phenotypic traits was observed in the hypomethylated population. Distinct DNA methylation patterns were detected using Methylation Sensitive Amplification Polymorphism (MSAP) in early flowering, late flowering, and small rosette diameter lines. The inheritance of methylation profiles were confirmed by bisulfite sequencing of targeted regions. The inheritance study following early flowering, late flowering, and late stolon emergence time variants through meiosis were demonstrated. It revealed early flowering was stably inherited for at least five meiotic generations but the late flowering phenotype reverted back to the control after two generations. Furthermore, the clonal transmission of the early flowering trait to daughter plants was also confirmed through mitosis. Characterization of the fifth generation (H4S13) early flowering lines was performed using high-resolution methylation combined with RNA-Seq. Differentially expressed genes involved in the flowering time pathway were detected, especially those involved in the photoperiod pathway. The up-regulation of downstream genes in flowering pathway NUCLEAR FACTOR Y, SUBUNIT B2 (NFYB2), FLOWERING LOCUS T (FT, the highest fold change), FRUITFULL (FUL) and SEPALLATA3 (SEP3) likely contributed to the floral transition. Significantly different CG methylation levels of the FT gene and coding sequence regions were observed. The overall DNA methylation rate at CG (63%), CHG (36%), and CHH (1%) sites based on single base resolution is first reported in strawberry. In addition, a novel MSAP method was established using the isoschizomers Tfi I / Pfe I to detect DNA methylation at CG, CHG, CHH sites based on restriction sites. Further application of this method into the early flowering lines found the maintenance of symmetric CG, CHG methylation over meiotic generations. This project for the first time developed a hypomethylated F. vesca population, comprehensively studied DNA methylation variation of quantitative traits, and the inheritance of these traits through meiosis and mitosis. These data add to the growing evidence that altering epigenetic variation can be a mechanism for generating increased phenotypic diversity and can be subject to selection. This research indicates the potential of crop improvement through epigenetic modification without changing DNA sequence, avoiding the issue of Genetically Modified Organisms.