Anthocyanins are important metabolites that provide a red or blue–purple hue to plants. The biosynthesis of these metabolites is mainly activated by the MYB-bHLH-WD40 (MBW) complex and repressed by a wide variety of proteins. Studies have shown that MYB activators activate MYB repressors to balance anthocyanin biosynthesis. However, there is a scarcity of studies investigating this mechanism in grapes. To explore the transcription factors involved in the regulation of anthocyanin biosynthesis, we reanalyzed the RNA-seq database for different developmental stages of ‘Muscat Hamburg’ berries, and the R2R3-MYB gene, annotated as VvMYB3, was screened. Our study revealed the anthocyanin content of the grape cultivar ‘Y73’ was higher than that of its parental cultivar MH, and the putative repressor VvMYB3 was found to be highly expressed in ‘Y73’ by qRT-PCR. The calli transgenic assays demonstrated that the repressive activity of VvMYB3 was conferred by the bHLH-binding motif, as well as by the C1 and C2 motifs. Yeast hybridization and chip-PCR assays revealed that VvMYB3 could repress anthocyanin biosynthesis by competing with VvMYBA1 to bind to VvMYC1 and promoting histone deacetylation of VvUFGT via the C2 motif. However, the expression of VvMYB3 was activated by VvMYBA1, which forms a negative feedback regulatory loop to modulate anthocyanin accumulation. In addition, we found a 408-bp repeat tandem sequence insertion in the VvMYBA1 promoter region of ‘Y73’ by sequencing. The GUS activity analysis showed that this sequence enhanced the expression of VvMYBA1 and led to an excessive accumulation of anthocyanins. Overall, our results provide insights into the anthocyanin activator–repressor system in grapes that prevents overaccumulation of anthocyanins.

Grape berry shape is an important agricultural trait. Clarifying its genetic basis is significant for cultivating grape varieties that meet market demands. However, the current study by forward genetics has not achieved in-depth results. Here, a high-density map was constructed to identify quantitative trait loci (QTLs) for berry shape. A total of 358709 polymorphic SNPs were obtained using whole-genome resequencing (WGS) based on 208 F₂ individuals derived from round grape ‘E42-6’ and oblong grape ‘Rizamat’. The 1635.65 cM high-density map was divided into 19 linkage groups with an average distance of 0.37 cM. Using this map, three significant QTLs for fruit shape index (ShI: ratio of berry length to berry width) identified over three years were mapped onto LG4 and LG5, including one stable QTL on Chr5 with the genomic region of 0.47–1.94 Mb. Combining with gene annotation and expression patterns based on RNA-seq data from two contrasting F₂ individuals with round and oblong berry (their average ShI was 1.89 and 1.10, respectively) at four developmental stages, four candidate genes were selected from the above QTLs. They were mainly involved in DNA replication, cell wall modification, and phytohormone biosynthesis. Further analysis of RNA-seq data revealed that several important phytohormone synthesis and metabolic pathways were enriched based on differentially expressed genes (DEGs), which was consistent with the results of QTL mapping for genes related to plant hormone biosynthesis in the F₂ population. Furthermore, a comparison of plant hormone content showed that there were significant differences in IAA and tZ content between the two contrasting F₂ individuals at different developmental stages. Our findings provide molecular insights into the genetic variation in grape berry shape. Stable QTLs and their tightly linked markers offer the possibility of marker-assisted selection to accelerate berry shape breeding.

The aim of this study was to characterize the changes in berry anatomy during the development of grape (Vitis vinifera L., and Vitis vinifera × Vitis labrusca) with different shapes. Paraffin sectioning was used to examine the structural parameters of the cells. The results revealed that, with the development of berries, the transverse and longitudinal diameters of the flesh cells gradually increased, revealing certain regularity. However, the transverse and longitudinal diameters of the epidermal and sub-epidermal cells were different between varieties, reflecting the specificity of the varieties. The growth of the transverse and longitudinal diameters of the berries was found to be completed in the early stage of development. A combination of correlation analysis and size analysis for each cell layer revealed that, due to the small cross-sectional area of the epidermal and sub-epidermal cells, the influence of these cells on the transverse and the longitudinal diameters of the berries would be small. In conclusion, the longitudinal and transverse diameters of the grape berries were mainly determined by the longitudinal and transverse diameters of the flesh cells. The different shapes of the grape berries could mainly be attributed to the different growth rates of the flesh cells in the longitudinal and transverse directions. These different rates of growth led to different lengths and widths of the berry.