Promoter Variation Analysis Of Key Gene MdPG1 Related To Texture Of Apple Fruit And Its Upstream Regulation

Apple（Malus domestica Borth.）is an important domestica tree that widely cultivated all over the world.China has the largest apple production worldwide in terms of area and yield.However,most of the main early-ripening apple cultivars soften and degrade easily during storage,which affects their economic value.Therefore,in-depth research on the molecular mechanism of fruit softening is needed to develop efficient molecular markers for the selection and breeding of storage-resistant early-ripening apple cultivars.In this study,apples with different softening rates were used as experimental materials.Through transcriptome analysis,multiple candidate genes related to apple softening were screened.On this basis,the promoter variation of candidate gene MdPG1（POLYGALACTURONASE1）and its upstream regulatory mechanism were studied,and the molecular mechanism by which the key SNP and lncRNA_PG1on MdPG1 promoter affect MdPG1 expression was clarified.The main results are as follows:1.We identified candidate genes responsible for fruit softening in apple.We evaluated the fruit firmness and ethylene production rates of 60 apple cultivars during shelf-life.There was a significant positive correlation between ethylene-production rates and fruit-softening rates.Five candidate genes related to apple softening were screened through transcriptome analysis,including MdACS3a,MdPG1,CYP707A（MD03G1088100）,MdMADS4 and SAUR（MD10G1060800）.2.We investigated the promoter variation and clarified the molecular mechanism by which the key single-nucleotide polymorphism on the MdPG1 promoter affect MdPG1expression.We identified variations in the 2.5 kb region upstream start codon of MdPG1by comparing the sequences of four apple cultivars.These variations included 2 structural variations,2 indels,and 39 SNPs in the promoter sequences of MdPG1.One SNP(SNP^C/A)identified above was within an ERF binding element.The SNP genotype is associated with fruit firmness and MdPG1 allele-specific expression level.Allele-specific expression of MdPG1 is regulated by MdCBF2,a member of the AP2/ERF transcription factor family.The promoter containing the ERF binding element with SNP^A,rather than the SNP^C,could be strongly bound and activated by MdCBF2 as determined by yeast-one-hybrid and dual-luciferase reporter assays.A cleaved amplified polymorphic sequence（CAPS）maker was developed based on the SNP,and the marker that based on a causative variant would be useful for selection of breeding seedlings with good fruit firmness before fruit production.3.We clarified the molecular mechanism by which the lncRNA_PG1 on the MdPG1promoter affect MdPG1 expression.We performed sequencing analysis of the MdPG1promoter region in the eight apple cultivars（‘Meiba’,‘Huahong’,‘Huaxing’,‘Huashuo’,‘Huaguang’,‘Hongcuibao’and‘Starking Delicious’）,and found a 1,335 bp insertion that was 2,356 bp upstream of the start codon of the larger MdPG1 fragment in‘Meiba’,‘Huaxing’,‘Huashuo’and‘Hongcuibao’;insertion sequence annotation identified long non-coding RNA（lncRNA）.Two transcript variants were identified as having 911 and 894bp of c DNA,and named lncRNA_PG1.The expression and distribution of lncRNA_PG1.Allele-specific expression analysis of MdPG1 in‘Huaxing’and‘Hongcuibao’showed that the MdPG1 allele containing lncRNA_PG1 expressed less than 8%of the gene transcript.Hybrid varieties containing homozygous lncRNA_PG1 maintained greater fruit firmness than those without lncRNA_PG1 because MdPG1 expression is lower in the homozygous lncRNA_PG1genotype.Luciferase transient expression assay in tobacco leaves showed that luciferase activity was reduced by fusing lncRNA_PG1 to MdPG1 promoter driving luciferase coding sequence.However,this reduction of luciferase activity was not detected when lncRNA_PG1was separated from MdPG1 promoter by using two different constructs.Histochemical GUS staining and fluorometric assays indicated that lncRNA_PG1 reduced the promoter activity of Nt CHLH（Mg-chelatase H subunit）in stable transgenic tobacco plants when the lncRNA and promoter were fused within one gene construct.The data presented in this study suggest that lncRNA_PG1 acts as a cis-regulator.DNA methylation analyses showed that the positions of CHH methylation in MdPG1 and MD10G1179400 promoters exhibited obvious differences between‘Huahong’and‘Huashuo’.These data suggest hat lncRNA_PG1might repress MdPG1 expression by changing CHH methylation locations,and might repress MD10G1179400 expression by altering methylation locations as well as increasing CG and CHH methylation level.In summary,we investigated the promoter variation and upstream regulation of MdPG1,and clarified the molecular mechanism by which the key single-nucleotide polymorphism and lncRNA_PG1 on the MdPG1 promoter affect MdPG1 expression.The result provides new understanding on plant lncRNA function and the regulation of fruit softening.