| Nanfeng tangerine is one of the famous local cultivars of China.It has been cultivated for more than 1300 years.During the natural evolvement and culture,Nanfeng tangerine group and Nanfeng guangju group were formed.The largest cultivation area in production is the small-fruit strain of Nanfeng tangerine group,followed by Miguang of Nanfeng guangju group.At present,it has significant difference on fruit quality of Nanfeng tangerine group.It shows that mastication of Nanfeng tangerine group gets inferior,which serious impacts on it′s commodity performance and economic value.The fruit quality of Miguang is excellent,while the Miguang/trifoliate orange tree is upright and fruit setting rate is low which limiting its popularization and application in production.In this study,the fruit of small-fruit strain of Nanfeng tangerine group（NM）was used as the experimental material,and Shatang tangerine fruit（ST）was used as the control.Transmission electron microscopy was used to observe the ultrastructure of cell wall in the segment membrane.The expression levels of the family members of the key enzyme genes on cell wall metabolism were detected by q RT-PCR.To find the key candidate genes,the correlation analysis was conducted based on the content of cell wall components and the expression levels of key enzyme genes.The full-length ORF sequences of key candidate genes were cloned,sequenced and bioinformatics analyzed to provide the basis for gene function verification,texture quality control and breeding using genetic engineering technology.At the same time,we compared the changes of phenotypic characteristics,phytohormone contentsand carbohydrate contents among trifoliate orange（TO,weak growth rootstock）,Flying dragon trifoliate orange（FD,weak growth rootstock）and Carrizo citrange（CC,vigorousrootstock）.The transcriptional regulation mechanism of dwarf characteristics of FD was revealed usingthe full-length transcriptome analysis.Miguang grafted on the CC,TO and FD rootstockswere obtained and used as experimental materials.The growth morphology,anatomical characteristics,contents of phytohormone and carbohydrate were studied.Genome-wide DNA methylation sequencing and transcripomics were used to reveal key genes/pathways of methylation-mediated dwarfing traits,and to elucidate the dwarfing effect and epigenetic regulation of Miguang/FD.The main results were as follows:1.The ultrastructure of cell wall in segment membrane was related to the mastication of NM fruit.Compared with NM,the segment membrane of ST was transparent,the middle layer of cell wall composed of pectin was almost invisible,and part of the cell wall was degraded.2.Four candidate genes for cell wall metabolism of NM segment membrane were identified and the sequences were analyzed by bioinformatics.BGL42,BGL46,EG4 and C4H were identified as the key candidate genes for cell wall metabolism in NM according to the correlation analysis between the expression levels of genes related to cell wall metabolism and the contents of cell wall components during fruit development.3.Compared with CC,FD and TO rootstocks had dwarfing characteristics in terms of phenotypic characteristics,phytohormone contents and carbohydrate contents in stem.Compared with TO,FD had more obvious dwarfing characteristics.The phenotypic observation revealed that the plant height,leaf size and thickness,plant weight,and internode length were reduced in dwarfing rootstocks.Moreover,the contents of IAA,t Z and ABA,were higher in FD and TO rootstocks,whereas the GA3content was higher in the CC rootstocks.（IAA+t Z+GA3）/ABA ratio in FD and TO rootstocks were lower than CC.The carbohydrate contents,including sucrose,fructose,glucose,starch,and lignin significantly decreased in both the FD and TO rootstocks.Compared with TO,FD had smaller leaf size,biomass,leaf area,leaf thickness,and spongy tissue thickness,and theinternodes length was basically the same while the internodes number was significantly fewer.4.Nineteen candidate genes related to dwarfing traits in FD were identified.Full-length transcriptome analysis showed that phytohormone metabolism and signal transduction in dwarf rootstocks were disturbed,and genes such as Aux/IAA,SAUR,GH3,GA2ox,GID1 and DELLA were closely related to changes in endogenous phytohormone content.Our results also demonstrated that the SUS,INV,FRK,TPS and AMS genes related to sugar degradation,and EG,BGL,XYL,XGOAT and XTH genes related to cellulose and hemicellulose hydrolysis were activated,whereas PAL,CAD and COMT genes involved in lignin biosynthesis were reduced in the FD and TO rootstocks.Moreover,many regulators,including TFs,lncRNAs,and AS events involved in the synergistic regulation of stem elongation and development in the FD and TO rootstocks were identified.5.Compared with MG/CC,MG/FD showed dwarfing characteristics in terms of growth morphology,anatomical characteristics,contents of phytohormone and carbohydrate.Results showed that the plant height,leaf thickness,leaf sponge tissue thickness and leaf photosynthetic performance of MG/FD and MG/TO plants were lower than those of MG/CC plants.In addition,the MG plants grafted on the dwarfing rootstocks,especially on the FD,accumulated more sucrose,fructose and glucose,but less lignin and cellulose in theirleaves.The determination of phytohormone revealed that IAA and ABA concentrations were significantly higher,while GA3and t Z concentrations were significantly lower in the MG young stems of MG/FD and MG/TO than those of MG/CC plants.6.Total of 934 differentially expressed genes（DEGs）were identified in the MG young stems between MG/CC and MG/FD by transcriptome analysis.KEGG analysis showed that these DEGs were mainly enriched in metabolic pathways,secondary metabolite synthesis,starch and sucrose metabolism,phytohormone synthesis and signal transduction pathways.7.Dwarfing traits of MG/FD were associated with low DNA methylation levels in MG stems.The DNA methylation of young stems in MG/CC and MG/FD was carried out by whole genome bisulfite sequencing（WGBS）.We found that the whole genome DNA methylation level and methylation rate of different regions in MG/FD were lower than those in MG/CC plants.A total of 2538 differentially methylated cytosines（DMCs）and794 differentially methylated regions（DMRs）were screened.8.It was found that SAC9,LBD19,CYCD1-1 and LAC17 were the key candidate genes for regulating methylation.Integrated methylation and transcriptome analysis unveiled 13 genes exhibited differentially both in methylation level and gene expression between MG/CC and MG/FD plants.Among these genes,the predictive functions of phosphoinositide phosphatase gene SAC9（Ciclev10018900m.g）,G1/S-specific cyclin gene CYCD1-1（Ciclev10026166m.g）,laccase gene LAC17（Ciclev10011362m.g）and LBD19 gene（Ciclev10022291m.g）were directly related to plant growth and development.These genes may be involved in the methylation regulation of dwarfing Miguang mediated by FD rootstock.In conclusion,the ultrastructure of cell wall in segment membrane was related to the mastication of NM fruit.BGL42,BGL46,EG4 and C4H were identified as the key candidate genes for cell wall metabolism in NM.FD had dwarfing traits in terms of phenotypic characteristics,phytohormone contents and carbohydrate contents in stem.Aux/IAA,SAUR,GH3,GA2ox,GID1,DELLA,SUS,INV,FRK,TPS,AMS,EG,BGL,XYL,XGOAT,XTH,PAL,CAD and COMT were candidate genes related to dwarfing traits of FD rootstock.FD rootstock had dwarfing effect on MG,which related to low methylation levels of DNA,SAC9,LBD19,CYCD1-1 and LAC17 were the key candidate genes. |
