Transcriptome Changes And Genome-wide DNA Methylation During Fruit Development And Ripening In Fragaria Vesca

Fruit ripening is a highly complex and synergistic biological process.The expression of genes related to ripening is regulated by various factors such as hormones and environmental factors.DNA methylation is one of the important epigenetic modifications in plant,which plays an important role in plant growth,morphogenesis and other aspects.However,there are few studies on the effect of DNA methylation on the development of non-climacteric fruits such as strawberries.This study provided a description of the transcriptomic changes during fruit development and ripening in diploid forest strawberry,along with a dynamic view of the gene expression difference in the five development stage.Moreover,we also analyzed the whole genome DNA methylation during strawberry fruit ripening.The main results are as follows:1.We compared the physiological characteristics of the five stages,small white(SW),big white(BW),pre-turning(Pre-T),Pink,and Red in Fragaria vesca.With the ripening of strawberry fruit,the firmness of fruit decreased gradually in general,except for that in the Pre-T stage which dropped dramatically.However,the contents of anthocyanin and soluble sugar increased rapidly in the Pre-T stage,and peaked at the Red stage.We used paraffin sections to observe the tissue structure of the cells.The size of strawberry fruit cells and the intercellular space continuously increased from BG stage to Pink stage,and there was almost no contact between some adjacent cells from the Pre-T stage.Based on these analyses,we conclude that Pre-T can be defined as the transition stage from immature to mature of strawberry fruit.2.Through analysis of DNA methylation differences between strawberry fruit at the stage of SW and Red,we obtained a total of 3210 methylated differential genes,among which 1890 and 1320 genes were up-regulated and down-regulated,respectively.In the stage of fruit ripening,the level of DNA methylation did not change much during the period from the SW to Pre-T,but increased significantly in the red fruit stage.For various functional regions,the overall methylation level was expressed in the CG context as:repeat>intron>promoter>exon,which appears as repeat>promoter>intron>exon at CHG and CHH context.The DNA methylation of the CG context is seen to be preferential in the gene region;in the gene body(and in the upstream and downstream 2kb regulatory regions),the DNA methylation levels of the red fruits are the highest at the three context(CG,CHG,and CHH).The level of DNA methylation between the SW and the Pre-T is similar.3.By analyzing the fruit transcriptome sequencing data of five developmental stages of strawberry,we obtained 8,968 differentially expressed genes.The number of differentially expressed genes SW to Pre-T was the highest,with a total of 4001,which may reflect the complex regulation of multiple genes in the strawberry fruit from immature to ripening stage.Through further significant enrichment analysis,we found that during the ripening transition(BW vs.Pre-T),there was some relative up-regulation of differentially expressed genes involved in flavonol/anthocyanin,aroma,fatty acid biosynthesis,folate/Vitamin B6 metabolism,biotic stress,cell wall-related terms,and ABA synthesis,and a decrease in expression of genes involved in the photosynthetic apparatus,oxidation-reduction processes,and he expression of auxin and BR-related genes.4.The SW and Red stage transcriptome data and whole genome methylation data of strawberry fruit were combined and analyzed to find common genes that showed both significant expression and methylation differences.After comparison,a total of 665 genes that showed significant differences in expression and DNA methylation also occurred.The transcription level of 240 genes was down-regulated while the methylation level was increased,and the transcription of 92 genes increased while the methylation level decreased in the process of strawberry fruit ripening.After further analysis,these genes are mainly involved in cell wall changes,anthocyanin-related pathways and some hormone metabolism pathways.5.Through further analysis,we obtained three DNA methylation modification genes(FveMET1,FveDML1 and FveDDM1)that may play an important role in the regulation of strawberry fruit development,and constructed corresponding RNA interference vectors,followed by Agrobacterium tumefaciens-mediated strawberry genetic transformation.,Obtained 10 to 50 independent lines of strawberry RNAi transgenic plants of FveMET1,FveDMLl and FveDDM1.At present,we observed that the RNAi transgenic plants of FveDDM1 showed obvious deformities in the leaves compared to the control plants,with narrowed leaf margins and deeper nicks.