The Patterns And Functions Of DNA Methylation During Floral Bud Differentiation Of Salix Viminalis

Salix viminalis,Salicaceae shrub,has the properties of wide distribution,strong adaptability,fast growth and easy reproduction.The childhood of Salix viminalis is short,seedlings flower in 2 years,and cuttings flower in one year.The stage of flower bud differentiation lasts for more than 3 months.The gender in Salix viminalis is stable.The genome is only 470 M.It is an ideal material for studying the flower bud differentiation and sex determination mechanism of woody plants.In this study,the shoot tips of Salix viminalis in the vegetative growth stage,physiological differentiation stage and flower bud differentiation stage were used as study materials.The methylation pattern of the shoot tip genome of Salix viminalis in different development stages was obtained by whole genome methylation sequencing.Differential methylation regions related to flower bud development(FDMR)were obtained through comparing methylation data between developmental stages.Sex differential methylation regions(SDMR)were obtained through methylation data comparison between female and male.The transcriptome data was used to obtain the expression data of SDMR and FDMR-related genes,and the expression patterns of SDMR and FDMR-related genes were verified by q RT-PCR.Further,function of DNA methylation in fllower development and sexual differentiation was verified by treating the Salix viminalis individuals with methylation inhibitor 5-aza C.The main results are as follows:(1)In the vegetative growth stage(S1),physiological differentiation stage(S2)and flower bud differentiation stage(S3),the methylation levels of total cytosine sites in the stem tip of Salix viminalis were 6.51%,7.02% and 7.39% respectively,which showed a gradual increase trend;the methylation levels of the female stems of Salix viminalis were 7.32%,6.69 and 7.18%,respectively,which showed a trend of decreasing at first,and then increasing.(2)In female and male,the number of flower development related DMR genes with expression differences of |log FC| > 1 was 1401 and 1597,respectively.In hormone-related DMR genes,auxin-responsive genes ARF7 and ARF22 were up-regulated by DNA methylation in floral organ meristem differentiation and vegetative growth to reproductive growth respectively;ABA regulated the downstream region methylation level of MYB3 gene,and increased its expression,activated downstream CO,and promoted flowering.In carbohydrate-related DMR genes,the expression of ?-amylase genes AMY1 and AMY2 were up-regulated by intron methylation during flower bud differentiation,providing energy for differentiation of floral meristem;?-amylase gene BMY5 expression was up-regulated by DNA methylation during physiological differentiation stage and participated in floral transition;T6P synthase genes TPS9 and TPS6 were up-regulated by DNA methylation during physiological differentiation stage and flower bud differentiation stage,respectively.And both of them are involved in T6 P signaling pathway.The expression of GI,AGL19 and SOC1 in the photoperiod flowering pathway were also regulated by DNA methylation.(3)Through the correlation analysis between DNA methylation data and transcriptome data,the expression difference of 702 SDMR genes were found to reach |log FC| > 1.The hormonerelated SDMR genes mainly include auxin metabolism and transport genes,such as AM11,GH3,D6 PK,TMK1,etc.The sex differential expression of these genes mainly expressed in the vegetative growth stage and flower bud differentiation stage.The expression level was higher in male than that of female;the abscisic acid response and signal transduction genes EXO70B1,AHG1,and ALDH3H1 were significantly up-regulated in male individuals during S1,S2,and S3 stages,respectively.Among SDMR genes associated with carbohydrates,the starch biosynthesis gene AGP-L and the ?-amylase gene BAM1 were significantly lower in S1 and S2 stages of male than that in female;The expression level of ?-amylase gene AMY2 in male at S3 was significantly lower than that in female;the expression level of sucrose synthetic gene SUS5 in male at S2 was significantly higher than that in female.Among the SDMR genes involved in flower transformation and flower bud development,the expression of the FT-transport gene FTI1 in male at S1 stage was significantly higher than that in female;the expression of FT-binding protein FD in male plants at flower bud differentiation stage was significantly lower than female plant.In addition,five SDMR genes related to synthesis,processing and degradation of small RNA were screened,in which RDR1,DCL1,DCL2-1,and DCL2-2 were higher in male plants than in female,and UTR1 was lower in male plants than female.(4)The methylation inhibitor 5-aza C significantly reduced the level of DNA methylation in leaves of Salix viminalis at flower development stage,but had no significant effect on the level of DNA methylation in leaves during physiological differentiation stage.In female plants,5-aza C treatment reduced the content of starch,soluble sugar and reducing sugar in the leaves at physiological differentiation stage,and promoted the differentiation and growth of flower buds of female plants.In male plants,the leaf carbohydrate content in the physiological differentiation period was significantly increased by 5-aza C treatment,while the number and diameter of flower buds were significant reduced.In summary,DNA methylation participates in flowering transformation of Salix viminalis by regulating carbohydrate metabolism,expression of key genes in photoperiod pathways,expression of hormone response-related genes,and expression of key flowering genes.In addition,DNA methylation can also participate in the sex differentiation of Salix viminalis by regulating the expression of floral organ development genes and the expression of genes involved in the small RNA biosynthesis pathway.