QTL Mapping And Functional Analysis Of Candide Genes Related To Fruit Fructose Content On Apple Chromosome 11

The flavor of apple fruit is mainly determined by the content and proportion of soluble sugar and organic acid in the fruit.Fructose is the sweetest soluble sugar,whose sweetness is1.73 times that of sucrose and 2.34 times that of glucose.Therefore,it is of great significance to explore the accumulation and genetic regulation mechanism of fructose content and key genes for apple sweet quality breeding in apple fruits.In the present study,quantitative trait loci(QTL)controlling fructose content in apple were identified on LG(Link Group)11 with‘Honeycrisp’(high fructose)× ‘Qinguan’(low fructose)hybrid F1 fruits as materials combined with high density genetic linkage map.MdTrx m4,a candidate gene regulating fructose content in fruits,was screened by SNP annotation and transcriptome data,and its function was studied by genetic transformation and prokaryotic expression analysis.The main results were as follows:1.The fructose content in ‘Honeycrisp’ is significantly higher than that in ‘Qinguan’.Fructose content in the fruits of ‘Honeycrisp’ × ‘Qinguan’ hybrid F1 was determined and analyzed for two consecutive years.The results showed that the fructose content in the hybrid progeny was widely distributed and normal distribution,which was in line with the genetic characteristics of quantitative traits.And the average fructose content in two years was 46.54±6.75 mg/g FW and 56.25±8.82 mg/g FW,respectively.Based on QTL mapping analysis of apple fructose content,two QTL loci were located on ‘Qinguan’ LG11 and three QTL loci were located on ‘Honeycrisp’ LG11 in 2015 and 2016,among which there was a stable QTL overlap interval in ‘Honeycrisp’ LG11 for two consecutive years.2.There were 12 genes in the overlap interval of ‘Honeycrisp’ LG11,and 6 of the 12 genes had heritable amino acid variation on the exon analyzed by combining parental resequencing and SNP annotation.Further analysis showed that the expression level of candidate gene MD11G1224900 in young and mature fruits of ‘Qinguan’ was lower than that in ‘Honeycrisp’ based on the transcriptome data of ‘Qinguan’ and ‘Honeycrisp’ fruits during their development stage.The q RT-PCR results unveiled that the expression level of MD11G1224900 was positively correlated with fructose content in the flesh of ‘Qinguan’and ‘Honeycrisp’.3.MdTrx m4 was cloned from cDNA of ‘Qinguan’ and ‘Honeycrisp’.Sequence analysis showed that there were two haplotypes of MdTrx m4 in ‘Qinguan’ and ‘Honeycrisp’respectively,which were named as Q1-MdTrx m4,Q2-MdTrx m4,M1-MdTrx m4 and M2-MdTrx m4.Bioinformatics analysis showed that Q1-MdTrx m4 was significantly different from the other three haplotypes in physicochemical properties,hydrophilicity and secondary structure.4.Overexpression vectors for the four haplotypes and MdTrx m4 interference vectors were constructed respectively and instantaneously transformed into the calli of ’Wanglin’apple flesh.Fructose content was determined,which shown that the fructose content in the overexpressed Q1-MdTrx m4 apple calli had no significant change compared with control,but the fructose content in the other three haplotype transgenic calli all were increased.And Fructose content were significantly decreased in all the interference transgenic calli.5.Prokaryotic expression vectors of four haplotypes were successfully constructed and proteins were induced and purified.Protein solubility analysis showed that the inclusion bodies of protein Q1-MdTrx m4 were more than soluble proteins while the other three proteins were on the contrary under the same induction condition.