The Effect Of DNA Methylation During Fruit Ripening And Leaf-to-Callus Transition Of Woodland Strawberry

Strawberry,a perennial herb belonging to Fragaria of Rosaceae.Its fruit has the characteristics of bright color,soft and juicy,unique flavor and rich nutrition,which enjoy the reputation of "Queen of Fruit".Generally,cultivated strawberry(Fragaria ×ananassa)belongs to octoploid plant,which has short life cycle with early fruit set and huge economic benefits.China is the largest strawberry planting area in the world,and it is also a big consumer of strawberries.F.ananassa genome is large and complex,so it is relatively difficult to study.Woodland strawberry(Fragaria vesca)has a small genome and is the most dominant subgenome of cultivated strawberry.In addition,woodland strawberry also has a mature genetic transformation system.Therefore,It is an ideal material for studying cultivated strawberry,and it has gradually become one of the important model plants in the field of plant research as well as the model plant of Rsaceae.DNA methylation is one of the important epigenetic modification pathways.It can involve in the growth,development and morphogenesis of plants.However,there are still few studies on non-respiratory trophic plants such as woodland strawberry.In this study,the woodland strawberry was used as the research object,DNA demethylase Fve ROS1.2-RNAi transgenic plants were obtianed,and the phenotype observation and sequencing analysis of the DNA demethylase Fve ROS1.2-RNAi transgenic plants were performed.The role of DNA demethylase gene Fve ROS1.2 and DNA methylation modification in the development and ripening of woodland strawberry fruit was analyzed.Resulting that Fve ROS1.2 gene silencing causing bloom promoted of woodland strawberry,smaller fruit and fruit weight reduction.Whole Genome Methylation Sequencing(WGBS)and RNA-sequence(RNA-seq)analysis found that DNA methylation may affect strawberry fruit development by regulating cell division-related genes.Besides,Fve ROS1.2 gene silencing promoted callus formation,while DNA methyltransferase inhibitor 5-azacytidine(5-aza C)inhibited callus formation.WGBS and RNA-seq analysis found that DNA methylation may affect the dedifferentiation process by regulating the expression of key genes such as ARF-LBD,wound signaling and cell wall relaxation.The above research can provide material and data basis for exploring the role of DNA methylation in the process of woodland strawberry fruit development and callus formation.The main findings of this study are as follows:1.Fifteen independent strains of woodland strawberry DNA demethylase Fve ROS1.2gene silenced were obtained.Observation of the transgenic plants found that silencing the demethylase Fve ROS1.2 gene had no obvious effect on vegetative growth,but it would cause delayed flowering of plants and reduced fruit size.The specific manifestations are that the flowering is delayed for 14 days,the horizontal and vertical stems of the fruit are reduced,the fruit becomes smaller,the number of outer peel cell layers is reduced,and the fruit weight is reduced.The transcriptome data analysis of Fve ROS1.2 gene-silencing fruits showed that the down-regulation of the expression of cyclin(Cyc)and cycle-dependent kinase(CDK)related genes may be the cause of the smaller ROS1.2 gene-silencing fruits2.The study on the DNA methylation level of woodland strawberry fruits in the Green and Red stage found that the two ecotypes of Ruegen and Hawaii 4 have different dynamic changes in DNA methylation during fruit development and maturity.As the fruit matures,the DNA methylation level in Ruegen fruits increases,while the DNA methylation level in Hawaii 4 fruits decreases.The analysis of the gene region found that the DNA methylation level of the Ruegen gene region was up-regulated during fruit development and maturity,while the DNA methylation level of the Hawaii 4 gene region was down-regulated,which was consistent with the change trend of the whole genome methylation level.The analysis of transposons revealed that the DNA methylation levels of transposons in Ruegen and Hawaii 4 fruits were down-regulated as the fruits matured.The above results indicate that the DNA methylation changes of different ecotypes are diverse during strawberry fruit development and maturation.It can be seen that the overall DNA methylation change trend of the two ecotype fruits of Ruegen and Hawaii 4 is different during the development and maturation process,which may be caused by the different expression of the methyltransferase gene.In addition,through the correlation analysis between the DNA methylation level and the transcriptome expression level,it was found that in Ruegen and Hawaii 4,the expression level of cyclin(Cyc)and cycle-dependent kinase(CDK)genes and the DNA in their promoter regions.The methylation level is negatively correlated,and DNA methylation may affect the fruit size by affecting cell division-related genes.Maturation-related genes such as cytokinin response regulators(ARR),expansin(EXP),xyloglucan endotransgluco-sylase/hydrolase(XTH)and The expression levels of transcription factors such as WRKY and NAC are negatively correlated with the DNA methylation levels of their gene regions.It is speculated that DNA methylation may affect strawberry fruit ripening.3.It was found that the silencing of Fve ROS1.2 gene is beneficial to leaf dedifferentiation and callus formation,and reduces leaf browning.The whole genome methylation map of woodland strawberry leaves and callus was drawn and the changes of DNA methylation in the process of woodland strawberry leaves dedifferentiation and callus formation were analyzed based on WGBS.The results showed that the DNA methylation levels of CG,CHG and CHH in woodland strawberry leaves were 50.14%,24.44% and4.30%,respectively;the DNA methylation levels of CG,CHG and CHH in callus were53.86% and 25.77%,respectively And 3.66%.The DNA methylation level of callus in CG and CHG is higher than that of leaves,while in CHH sequence,the methylation level of DNA is lower than that of leaves.Further analysis and comparison showed that the m CG enrichment in the transposon region decreased slightly during the callus formation process,while the adjacent upstream or downstream regions increased.In contrast,the methylation enrichment levels of m CHG and m CHH in the transposon region and surrounding sequences were reduced.Five DNA methyltransferase genes were up-regulated during callus formation: Fve DNMT2,Fve DRM1.3,Fve CMT2,Fve CMT3.1 and Fve CMT3.2.The chromosome remodeling gene Fve DDM1,which maintains the DNA methylation level of the entire genome,is also slightly up-regulated.Transcriptome analysis showed that among31 differentially expressed genes related to callus formation and shoot regeneration during woodland strawberry dedifferentiation,the expression of the positive regulators Fve WOX14,Fve RAP2.6L,Fve E2 Fa,Fve PLT3/7,Fve PLT5,Fve WIND3,Fve WIND4,Fve CYCD3.1,and Fve CYCD3.3,etc was up-regulated,and the expression of negative regulatory factor Fve IAA14 was down-regulated.Combined with DNA methylation analysis,it was found that the expression changes of 8 genes including Fve PLT3/7,Fve WIND3 and Fve WIND4 are negatively correlated with DNA methylation changes,Indicated that these genes may play a regulatory role in the process of leaf callus formation through methylation-regulated gene expression.4.The DNA methyltransferase inhibitor 5-azacytidine(5-aza C)treatment of leaf explants can inhibit callus formation,showing the importance of DNA methylation for callus formation.Transcriptome analysis was performed on the woodland strawberry explants treated with 5-aza C for 0.5 and 3 days to investigate the potential mechanism of blocking DNA methyltransferase to inhibit strawberry leaf dedifferentiation.The results showed that compared with the callus formation process,some cell wall regulation related genes such as expansin(EXP),xyloglucan endoglucosylase(XTH),pectin lyase(PL)and pectin methyl esterase(PME)were differentially expressed,including The differential expression of some ethylene,gibberellin and growth hormone signal pathway genes such as Fve TINY2,Fve GASA1 and Fve IAA33 and transcription factors such as Fveb HLH and Fve WRKY.These results indicate that 5-aza C treatment might inhibited callus formation by down-regulating cell wall relaxation regulator genes,hormone signaling pathway genes,and some transcription factors homologous to organogenesis-related genes.