| ABSTRACTGrape berry skin is an important factor that affects commercial quality of grape and wine, and it is one of the most significant changes in fruit ripening process. The genes involved in anthocyanin biosynthesis in grapes are related to structural genes and regulatory genes. Recent literatures have indicated that white-skin grapes were the result of Myb A1 inserted into a retrotransposon Gret1 in an upstream coding region, and the Vvmyb A1 genotype determines berry skin color, and blocking the expression of this gene leads to the white berry phenotype. In the past, studies explored almost all of wild Vitis species that have black fruits; Among these, Vitis davidii is a wild Vitis species native to China, which has both white and pigmented-skinned berry genotypes. To uncover the molecular mechanisms responsible for the V. davidii white-fruit phenotype, we identified genetic relationships using SSR(simple sequence repeat) markers between white-skinned berry accession V. davidii and the other pigmented skin berry 40 accessions that originated from the southern region of China, and researched the Myb A1 alleles from pigmented and white-skinned cultivars of V. davidii to elucidate the molecular mechanisms driving phenotypic variation in grape skin color in V. davidii. We also performed a comparative analysis of anthocyanin profiles of grape berry skin, pulp, and seeds at different growth stages in V. davidii and control samples V. vinifera ‘Pinot blanc', ‘Pinot noir', ‘Muscat rouge', and ‘Muscat blanc' which were previously reported skinned mutants. The transcriptomic analysis was performed to compare the difference of transcriptional level in the black and white V. davidii, in order to find some key genes that are related to anthocyanin accumulation in V. davidii, and to find the reasons of the appearance of white V. davidii. The main results of this study were as follows:1. A total of 94 SSR markers were used for the analysis of the genetic relationship between white and pigmented V. davidii fruits. Genetic similarity was used for cluster analysis. The results of dendrograms showing the relationship according to the data obtained from SSRs showed that the V. davidii accessions and the control V. vinifera samples were classified into two main clusters using the genetic similarity coefficient. V. vinifera representatives were classified into three main clusters with the genetic similarity coefficient, which was interesting since the V. vinifera samples were collected from three areas. There was a high genetic similarity coefficient between the white-skinned fruit V. davidii ‘Baiciputao' and the pigmented ‘Hunanci', which belong to the same cluster, and in fact, were collected in the same region. Additionally, fluorescent markers SSR capillary electrophoresis was used to determine the molecules amplified. These data show that Baiciputao and Hunanci may have the same genetic background.2. According to the growth curves, samples were taken 40, 60, 80 or 90, and 100 or 120 days after anthesis(DAA) to identify the representative growth stages. The elementary composition of the anthocyanin profiles, non-anthocyanin phenolic contents, sugar and acid of grape berry skin, pulp, and seeds at different growth stages in V. davidii and control samples were analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry(HPLC-ESI-MS/MS), revealing divergent chemical profiles. The results showed that there were anthocyanins detected in grape berry skin and seeds from early growth stages to ripening stages,but no anthocyanins were detected in grape berry pulp at growth stages. Malvidin 3-O-glucoside(Mv G) was only present in pigmented grapes from samples obtained from veraison to maturity and was the most abundant anthocyanin identified during the ripening stages in pigmented grapes. However,there were no significant different of some anthocyanin between V. davidii white-skinned Baiciputao and color-skinned Hunanci, such as delphinidin 3-O-glucoside(Dp G). The PCA results showed that the main contributors to total variance were Mv G and Dp G. In addition, there were no significant different in non-anthocyanin phenolic contents, sugar and acid changes, and the antioxidant properties between colored- and white-skin grapes. These data provided supported for the conclusion that the skin color mutation leading to a white skin phenotype in V. davidii were related the major anthocyanins changes, such as Mv G.3. A PCR primer set was used to amplify the coding region between Myb A1 and Gret1 in V. viniferia. In both V. davidii and the control V. vinifera, the white-skinned grapes contained only Myb A1 a at the Myb A1 locus, while Myb A1 c was detected in the pigmented berry samples. Sequence analysis results revealed that the Myb A1 a gene was highly homologous among both pigmented and white-skinned V. davidii types and V. vinifera.4. To verify the gene expression differences of Baiciputao and Hunanci, the transcriptome of grape berry skin during growth stages were analyzed, the results showed that, although there were significant difference in the expression level of genes related to the anthocyanins accumulation between Baiciputao and Hunanci, there were no significant difference in functional category enrichment of gene ontology(GO). Moreover, the functional category enrichment of Kyoto Encyclopedia of genes and genomes(KEGG)analysis showed that the different expression genes were main related totaurine and hypotaurine metabolism pathway, phagosome pathway, and oxidative phosophorylation pathway. Relative expression profiles of structural genes and regulatory genes of grape berry skin, pulp, and seeds at different growth stages in V. davidii and control samples were analyzed by quantitative real-time PCR(q RT-PCR), the results showed that the expression of the regulatory gene Myb A1 in colored skin grapes was significantly higher than that in white skin grapes,especially after veraison. The expression of the structural genes UFGT, CHS, F3'H, F3'5'H, GST, and OMT were similar to that of Myb A1. The results of the correlations between anthocyanin content and the expression of the anthocyanin regulatory and structural genes showed that only Mv G in grape berry skin was positively related to the expression of the regulatory gene and structural genes. |
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