QTL Mapping Of Fructose Content And Functional Analysis Of Hexose Transporter MdHT2.2 In Apple

Sugar composition and content are the important indicators for apple fruit quality.Fructose(Fru),as the most sweet soluble sugar,accounts for about 50%of the total sugar content in apple,which plays a decisive role in fruit sweet quality.In this study,the hybrid population of‘Honeycrisp(HC,higher fructose content genotype)'בQinguan(QG,lower fructose content genotype)'was used as the material,combine with the high-density genetic linkage map,the quantitative trait loci(QTL)mapping was performed to find the major genetic regulation genes for fructose content and molecular markers in apple sweetness quality breeding.Moreover,the function of apple fruit hexose transporter Md HT2.2 in sugar accumulation was analyzed.The aim of this study was to explore the genetic control genes for fructose content and the regulation mechanism of fructose accumulation,and provide new insights and theoretical basis for sugar content quality improvement and genetic breeding.The main findings are as follows:1.QTL mapping for apple fruit sugar content,selection and functional analysis of fructose content regulation genes.1.1 QTL mapping for sugar content in apple fruit.With the hybrid population of‘HC'בQG'fruits as materials,the soluble solid content(SSC),fructose,glucose(Glc),galactose(Gal),sorbitol(Sor),sucrose(Suc)and total soluble sugar(TSS)conents were analyzed for two consecutive years.The results indicated that the content of different sugars showed normal distribution in two years.In 2015,the SSC was 8.3-17.4%Brix,the Fru,Glc,Gal,Sor,Suc and TSS contents were 28.26-65.92,16.36-76.65,2.02-9.02,0.49-15.63,2.61-48.82 and 86.83-172.54 mg/g FW,respectively.In 2016,the SSC was10.7-19.4%Brix,the Fru,Glc,Gal,Sor,Suc and TSS contents were 39.00-80.82,12.33-77.45,1.24-8.40,1.27-15.20,5.00-51.96 and 107.91-194.88 mg/g FW,respectively.The correlation analysis between sugar contents indicated that SSC and Suc,SSC and Sor,Glc and Gal,Sor and Suc showed significantly positive correlation,while Glc and Suc,Gal and Suc showed negative correlation.The QTL mapping for sugar content indicated that in the overlapping segments of the stably QTLs for two consecutive years,SSC is distributed on LGs 01,09 and 17 of‘QG';Glc content is distributed on LG 01 of‘HC'and LG 16 of‘QG';Suc content is distributed on LG 01 of‘HC';Fru content is distributed on LGs 01and 03 of‘HC',and the QTL-Fru-LG01 region has the highest contribution to fructose content.1.2 Selection and functional analysis of genetic control genes for fructose content in QTL-Fru-LG01 region on‘HC'.According to the genomic information,315 genes were found in this region,including 22 genes related to sugar metabolism,transport or transcription regulation,which contained seven tandems repeat Md SDH(sorbitol dehydrogenase)genes.RNA-seq and q RT-PCR analysis indicated that the expression of Md SDH2 increased at the late stage of fruit development in‘HC',but not siginficiant in‘QG',which was consistent with the changing pattern difference of SDH enzymes activity and fructose content during the‘HC'and‘QG'fruits development.In addition,the coding sequence of Md SDH2 is consistent in‘HC'and‘QG'.By cloning Md SDH2 gene in‘HC'and expressed in apple fruit calli,it was found that SDH enzyme activity increased by about 30%,fructose content increased by about 20%,sorbitol content decreased by about20%,while in the silent lines,the fructose content decreasesed but the sorbitol content increased.These results indicated that the expression difference of Md SDH2 in the QTL region on‘HC'LG 01 could control the fructose content in apple fruit.1.3 To investigate the reason why there were different expression patterns for Md SDH2 between‘HC'and‘QG'in developmental fruits,four haplotypes of the Md SDH2promoter were cloned from‘HC'and‘QG'respectively.The sequence alignment revealed that,in the two haplotypes of HC-Md SDH2 promoter region,there were 13 SNP sites,which contained six different transcription factor binding site.A specific mutantion in‘HC'called SDH2p-491(A/G)was selected,which is at the-491bp upstream from the start codon ATG of Md SDH2 gene.The EMSA,Dual-luciferase,CHIP-q PCR and GUS activity analysis showed that this SNP mutation affects the binding ability of Md ABI3,which is a transcription factor related to the ABA signaling pathway.While Md ABI3 could strongly bind to the haplotype of the HC-Md SDH2 promoter at SDH2p-491(A),but it slightly bind to the haplotype with SDH2p-491(G)genotype or the Md SDH2 promoter in‘QG'.The transient overexpression of Md ABI3 in‘HC'could significantly increase the expression of Md SDH2 and the content of fructose,but in‘QG',it was slight affected.Meanwhile,exogenous application of ABA can significantly induce the expression of Md ABI3,and up-regulate the expression of Md SDH2 in‘HC'or the hybrid progeny with A/G genotype,while it was not that obviously observed in‘QG'or the hybrid progeny with G/G genotype.This was consistent with the ABA content and the expression patterns of Md ABI3 and Md SDH2 during fruit development of‘HC'and‘QG'.These results indicated that the variation of SDH2p-491(A/G)site in the Md SDH2 promoter affects the binding ability of the ABA-inducible transcription factor Md ABI3,regulates the expression of Md SDH2 in the late stage of fruit development,and controls the fructose content.In addition,the analysis of the relationship between different genotypes of SDH2p-491(A/G)and the expression of Md SDH2,SDH enzyme activity and fructose content in hybrid progeny and cultivars revealed that this SNP site could be a marker for fructose content in molecular assisted selection breeding of domesticated apple.2.Functional analysis of the hexose transporter Md HT2.2 in the regulation of fructose content.After the phylogenetic analysis,transmembrane structure prediction,spatiotemporal expression analysis,subcellular localization,and yeast functional complementation experiments,the results showed that Md HT2.2 was located on the plasma membrane and highly expressed in parenchyma cells at fruit ripening stage.Meanwhile,it is a hexose/H~+symporter with high transport activity for fructose and glucose.In tomato,transgenic lines heterologously expressing Md HT2.2 showed dwarfing,enlarged fruits and more seeds.The hexose content in mature leaves of transgenic lines was lower than that of wild type,but the sucrose was higher.Moreover,there was no significant difference of hexose content in young fruit stage of all plant types,but the sucrose content increased significantly in transgenic lines.Moreover,the SSC,fructose and glucose content significantly increased in the mature fruit of transgenic lines,but the sucrose content significantly reduced.After the analysis of sugar metabolism-related enzyme activities and gene expression levels,it was found that the significantly increasing of CWINV(cell wall invertase)activity and the expression level of its coding gene Sl LIN5 in mature fruit of transgenic lines might be related to the sucrose content decreasing.In the Md HT2.2 overexpression line,by using CRISPR-Cas9 technology to knockdown sllin5,it showed that CWINV activity decreased,the sugar content change caused by Md HT2.2 overexpression recovered,and the sucrose content increased.These results indicated that hexose transporters could regulate the expression and activity of CWINV by regulating apoplastic hexose signals,thereby affecting sucrose transport,distribution and sugar content in fruit.Moreover,in different tissues and developmental stages,sugar metabolism and sugar signals are significantly difference.