Identification And Validation Of Genes Controlling Apple Fruit Acidity And Establishment Of The Genomic Selection Model

Malic acid(malate)is the most predominant organic acid,and plays an important role in apple fruit qulity.Therefore,analysis of genetic and molecular mechanism of apple fruit malate accumulation is fundamental for high-quality breeding and production of high quality fruits.To assess the genetic variation and determine major QTLs associated with malate content in apple fruits,we performed QTL mapping based on MapQTL and BSA-seq using two independent pedigree-based populations 'Jonathan' × 'Golden Delicious' and 'Zisai Pearl' × 'Red Fuji'.The variations and function of candidate genes from the major QTLs to control fruit malate content were further confirmed through a series of experiments,and the genomic selection model for fruit acidity was established.The main results are showed as follows:1.By analyzing the phenotypic data of individuals from 'Zisai Pearl' × 'Red Fuji' and its reciprocal cross,'Zisai Pearl' × 'Golden Delicious' and its reciprocal cross,and 'Jonathan' × 'Golden Delicious' in three year 2014-2016,we found that apple fruit acidity trait was quantitatively inherited and segregated extensively among individuals.The broad sense heritability of the five hybrid crosses were more than 80%,indicating that the variation of fruit acidity was determined mainly by genetic effect.2.Four major QTLs qtl08.1,qtl08.2,qtl08.3 and qtl16.1with stability over years and/or parental robustness were identified through MapQTL and BSA-seq approaches.Candidate genes were selected based on the annotation of the apple reference genome and re-sequencing database of the parents.The results show that MdMYB44,MdPP2CH,MdSAUR37 and MdALMTII were selected as candidate genes in locus of qtl08.1,qtl08.2,qtl08.3 and qtl16.1 separately.3.A previously developed dCAPS marker of MdALMTII was used to determine three genotypes(AA,AG,and GG)of MdALMTII in hybrids of 'Jonathan'× 'Golden Delicious' and 'Zisai Pearl' ×'Red Fuji',and Malus germplasm accessions.Genotypes of MdALMTII(AA,AG,and GG)were significantly associated fiuit malate content(P<0.01).Fruit malate contents of individuals with the genotype AG and GG were significantly higher than individuals with AA.4.A single nucleotide mutation A/G variant in MdPP2CH gene at position 802 of open reading frame(ORF)changed a ACG codon to GCG,and led to an amino acid substitution(threonine to alanine)at position 268(T268A).Genotypes of MdPP2CH(AA,AG,and GG)were significantly associated fruit malate content(P<0.001).Fruit malate contents of individuals with the genotype GG were significantly higher than individuals with AG,while individuals with the genotype AG were significantly higher than individuals with the genotype AA.When the amino acid changed from Thr to Ala,the phosphatases activity of MdPP2CH decreased significantly.The malate contents were significantly lower in overexpressing MdPP2CH transgenic calli lines than in WT,implying that MdPP2CH negatively regulated the accumulation of fruit malate.The malate contents in overexpressing MdPP2CH(268T)transgenic calli lines were significantly lower than in overexpressing MdPP2CH(268A)transgenic calli lines,implying that When the amino acid changed from Thr to Ala,the phosphatases activity of MdPP2CH declined.MdPP2CH interacted with MdVHA-A3,MdVHA-B2,MdVHA-D2 or MdALMTII and contributed to the decrease in malate contents via dephosphorylation of MdVHA-A1,MdVHA-B2,MdVHA-D2 and MdALMTII,possibly leading to a decline in malate transport into vacuole.5.A 36-bp insertion(at position-639 bp upstream ATG codon)in the promoter region were detected in MdSAUR37 gene.Genotypes of MdSAUR37(?,??,?)were significantly associated fruit malate content(P<0.001).Fruit malate contents of individuals with the genotype ? were significantly higher than individuals with ??,while individuals with the genotype ?? were significantly higher than individuals with the genotype ?.The expression of MdSAUR37exhibited a close correlation(R2=0.7792,P<0.001)with fruit malate content in different genotypes in ripened fruit.GUS histochemical assays and GUS/GFP reporter expressions suggested that the 36-bp insertion enhanced the basal promoter activity.MdSAUR37 interacted with MdPP2CH and inhibited the phosphatases activity of MdPP2CH.The MdPP2CH activity was reduced by about 35%by MdSAUR37.6.In the promoter region,a SNP(A/G variant at position-1010)were detected in MdMYB44 gene.Genotypes of MdMYB44(AA,AG,and GG)were significantly associated fruit malate content(P<0.001).Fruit malate contents of individuals with the genotype GG were significantly higher than individuals with AG,while individuals with the genotype AG were significantly higher than individuals with the genotype AA.7.Close association analysis between fruit malate contents and genotypes of MdSAUR37/MdPP2CH/MdALMTII/MdMYB44 variations were analysed.The ratios between allelic variations were estimated by using observed phenotype values,and the genotype values(GV)were tentatively assigned to the genes.We then proposed that a predicted phenotype values(PPV)should be the summation of the multiplication of GVs:PPV= GV(MdSAUR37)× GV(MdPP2CH)×GV(MdALMTII)+ GV(MdMYB44)+ Calibration coefficients(1).The PPVs were significantly correlated with the observed phenotype values of fruit malate content(p<0.001).