Comparing RNA-Seq And DNA Methylation Patterns In Apple (Malus Domestica Borkh.) Buds With Different Flowering Capabilities And Expression Analysis Of Candidate MdCDF Genes

�Fuji'is the main cultivated variety of apples in China,it has few and inferior flower buds,which limits the production of apples.DNA methylation,a chromatin modification,plays key roles in various biological processes in plants,such as growth,development and flower induction.But the whole genome DNA methylation levels and the regulatory mechanisms for the floral induction and transformation of DNA methylation in apple still unclear.Therefore,it is of great theoretical and practical significance to explore the expression patterns,methylation levels and to reveal the relationship between gene methylation and expression levels of apple buds with different flowering capabilities.In addition,it will explain the role of DNA methylation and enrich the basis theory of apple flower induction.In this study,�Nagafu No.2'buds having different flowering capabilities were selected as materials?Ab:axillary buds with no flowering;Lb:long-shoot buds with a low flowering rate;and Sb:spur buds with a higher flowering rate?.The morphological and physiological characteristics of these materials were compared.Then we determined the whole genome DNA methylation and the regulatory relationships with related genes by using RNA-seq and WGBS.The candidate key gene MdCDF2 with different DNA methylation and gene expression level was screened,and we analyzed the regulation mechanism involving with DNA methylation of floral induction in apple.The main findings are as follows:1.Comparative analysis of transcriptome.The RNA-seq libraries of buds with different flowering capabilities?Ab,Lb and Sb?were constructed,the morphological and physiological characteristics of different buds were analyzed,and it was found that the contents of IAA,BR,GA and CTK of Sb were significantly lowest compared with those of Ab and Lb,but the contents of ABA and carbohydrate showed the opposite result.The number of DEGs that were significantly different between different bud types was investigated.We found 674,750 and 524 DEGs that showed differential expression levels in Sb_vs_Lb,Sb_vs_Ab and Lb_vs_Ab,respectively.We identified DEGs,such as SUS5/6,VAS,TPP1 and TPR1,which are involved in carbohydrate metabolism,NCED1,CCD4 and PYL4,which are involved in hormone,that were up-regulated between in the Sb compared with in the Lb and Ab.And SPL13,SOC1 and AP1 which are involved in flowering were significantly differentially expressed.In addition,some TFs,including WOX1,WRKY13 and HSP70 genes,were up-regulated in the Sb compared with in the Lb and Ab.2.Comparative analysis of DNA methylation.The whole genome BS-seq libraries of buds with different flowering capabilities were constructed,and the whole genome cytosine methylations were identified.We analyzed the model of whole genome DNA methylation.We found that CHH methylation was highest in Ab?69.16%?while CG and CHG methylation was highest in Lb?16.71%and 15.42%,respectively?.Using a comparative analysis of DNA methylation levels in different genomic regions,we found that there were significant differences in DNA methylation levels in different gene regions among apple buds with different flowering capabilities.In detail,the DNA of Lb was relatively hypermethylated compared with that of Sb,which had a higher flowering rate,and this included the CG,CHG and CHH contexts of the promoter,5?UTR,exon,intron and 3?UTR regions.A similar result was seen in the comparison of Sb and Ab,indicating that DNA methylation in Ab,which did not undergo flowering,were relatively hypermethylated compared with the Sb.In addition,differential methylation regions were identified in the three comparison groups,the IGV snapshots of the representative hypo-and hypermethylated DMRs in apple buds with different flowering capabilities were shown,in which CDF2 gene was hypermethylated in the Sb compared with in the other buds.3.Correlation analysis between DNA methylation and gene expression.We classified genes into the following four quartiles based on their FPKM values:no?none:FPKM<0.1?,lower?1st:0.1<FPKM<10?,middle?2nd:10<FPKM<100?and higher?3rd:FPKM>100?expression,and methylated genes were further divided into five quintiles based on promoter and gene-body methylation levels:the 1st group had the lowest and the 5^th group had the highest DNA methylation level,as follows:1st:methylation level<20%;2nd:20%<methylation level<40%;3rd:40%<methylation level<60%;4th:60%<methylation level<80%;and 5th:methylation level>80%.We found that the genes with highest methylation level?5th group?in the gene-body region had the lowest expression levels in buds with different flowering capabilities,suggesting that gene-body methylation is negatively correlated with gene expression.However,contrary to the above results,the genes with the lowest methylation level?1st group.?in the promoter region had the lowest expression levels in buds,indicating a positive correlation between gene methylation and expression in the promoter regions.4.Identification and expression analysis of candidate gene family MdCDF and the functional study of MdCDF2.Eleven members of the family were identified in the apple genome,and the physical and chemical properties,evolutionary analysis,gene structure were carried out.The expression patterns of all MdCDF genes were determined during flowering induction by RT-qPCR.The results indicated that MdCDF2 and MdCDF6 in response to sucrose treatment,and the expressions were reduced.Subcellular localization showed that they were in the nucleus,and the Arabidopsis of overexpression MdCDF2 appeared late flowering.