*3.2. Differentially Expressed Gene (DEG) Analysis*

Through the differential expression analysis of the RNA-Seq data, 3124 DEGs were selected between It11 wpf ('Italia' grape skin samples at 11 weeks post-flowering) and It10 wpf. Compared to Be10 wpf, a total of 2707 DEGs were selected in Be11 wpf. In addition, 1766 DEGs were found between Ma11 wpf and Ma10 wpf, with 1716 DEGs between Fm11 wpf and Fm10 wpf. Rb11 wpf showed a total of 1640 DEG compared with Rb10 wpf, and Rr11 wpf showed 1579 DEGs compared with Rr10 wpf. The number of

upregulated DEGs at the véraison stage (10 wpf to 11 wpf) was greater than the number of downregulated DEGs among the three white varieties of 'Italia', 'Muscat of Alexandria', and 'Rosario Bianco', while in red-colored varieties, 'Benitaka' and 'Flame Muscat' both showed lower upregulated DEG numbers at the véraison stage. Be10 wpf showed 1731 DEGs compared to It10 wpf, Fm10 wpf displayed 2790 DEGs compared to Ma10 wpf, and Rr10 wpf had 2962 DEGs compared to Rb10 wpf. For bud sport varieties in 'Benitaka', 'Italia', 'Flame Muscat', and 'Muscat of Alexandria', more upregulated DEG numbers were found at 10 wpf. Be11 wpf had a total of 2074 DEGs compared to It11 wpf, while 2000 DEGs were screened between Fm11 wpf and Ma11 wpf. Rr11 wpf had 3282 DEGs compared to Rb11 wpf. Among three comparisons of 'Benitaka' versus 'Italia', 'Flame Muscat' versus 'Muscat of Alexandria', and 'Rosario Rosso' versus 'Rosario Bianco', more downregulated DEG numbers were found at 11 wpf (Table 2).


**Table 2.** The numbers of DEGs among difference comparison groups.

#### *3.3. Correlation Analysis among Each Sample*

The Pearson correlation coefficient between It10 wpf and It11 wpf was close to 1, and It10 showed a positive correlation with It11. The Pearson correlation coefficients between It11 and Ma11 and between It11 and Rb11 wpf were also close to 1. The three white varieties of 'Italia', 'Muscat of Alexandria', and 'Rosario Bianco' showed good correlation (>0.8) at 11 wpf as well. The correlation coefficients between Ma10 wpf and Ma11 wpf and between Rb10 wpf and Rb11 wpf were close to 1, with 'Muscat of Alexandria' and 'Rosario Bianco' closely correlated. The correlation between the three red varieties of 'Benitaka', 'Flame Muscat', and 'Rosario Rosso' was low between 10 wpf to 11 wpf (Figure 3).

#### *3.4. Gene Expression Level of VvMYBA1 in Berry Skins*

The log2FC value was used to compare the expression levels of *VvMYBA1* in the comparisons of the GC10\_vs\_RC10 group and the GC11\_vs\_RC11 group. The results showed that log2FC >7, which means that the expression levels of the *VvMYBA1* gene in the three red varieties were much higher than those in three green varieties. In the comparison of the GC10\_vs\_GC11 group, the log2FC value was only 1.26, and *VvMYBA1* just reached the differential expression level (if the screening parameter was log2FC > 1.5, then it was not significant). In the comparison of the RC10\_vs\_RC11 group, the log2FC value was 1.95, and the expression of the *VvMYBA1* gene was significantly different (Figure 4).

**Figure 3.** Correlation heatmap of six varieties (three groups).

**Figure 4.** Differences in *VvMYBA1* expressions in red and white grape berry skins. 'GC' represents three green cultivars; 'RC' represents three red cultivars. a, b represent the significant level between the data (*p* < 0.05).

#### *3.5. Anthocyanin-Synthesis-Related Gene Expression Analysis*

Charenone synthase (CHS) is the first key enzyme of the flavonoid pathway. The gene expression in 'Benitaka' was higher at 10 wpf than at 11 wpf, and the gene expression level of Rr was lower at 10 wpf than 11 wpf. The expression level of the CHS-encoding gene *VvCHS* was significantly different at the véraison stage for 'Benitaka' and 'Rosario Rosso' compared with other varieties. The TPM values of *VvCHS* in berry skins at the véraison stage during the transition period of 'Benitaka' were higher than those of 'Italia' (Figure 5A). Chalcone isomerase (CHI) catalyzed the isomerization of chalcone rings to form colorless flavonoids, and there was no significant difference in the expression of the coding gene *VvCHI* between 10 wpf and 11 wpf for each cultivar (Figure 5B). Flavanone 3-hydroxylase (F3H) is one of the key enzymes in the biosynthetic pathway of anthocyanins, while F3H, F3'H, and F3 ,5 H participate in the regulation of two branches of anthocyanin biosynthesis and the F3 H-controlled pathway for the synthesis of red anthocyanins. F3 ,5 H, on the other hand, regulates the synthesis of blue-violet delphinidin. The expression level of the F3 H-encoding gene *VvF3 H* was low in each sample, and there was no significant difference between 10 wpf and 11 wpf (Figure 5C). The F3 ,5 H-encoding genes of *VvF3* and *5 H* were not expressed in It 10 wpf and Rr10 wpf, and the expressions of *VvF3* , *5 H* in the grape berry skins of the two mutated red varieties, 'Benitaka' and 'Flame Muscat', were higher than in 'Italia' and 'Rosario Bianco' (Figure 5D). In addition, the expression level of *VvF3H* in 'Benitaka' was obviously higher than that in 'Italia', and the expression of the F3Hencoded gene *VvF3H* at 10 wpf and 11 wpf for each sample was very low and displayed no difference in each cultivar (Figure 5E). The expression level of the FLS-encoding gene *VvFLS* showed greater variation in the skin of the 'Benitaka' during véraison.

**Figure 5.** Anthocyanin synthesis structure gene and regulatory gene expression analysis of six varieties. (**A**) *CHS*, chalcone synthase; (**B**) *CHI*, chalcone isomerase; (**C**) *F3 H*, flavonoid 30-hydroxylase; (**D**) *F3 5 H*, flavanone3 ,5 -hydroxylase; (**E**) *F3H*, flavanone 3-hydroxylase; (**F**) *FLS*, flavonol synthase; (**G**) *DFR*, dihydroflavonol 4-reductase; (**H**) *LDOX*, leucoanthocyanidin dioxygenase; (**I**) *LAR*, leucoanthocyanidin reductase; (**J**) *UFGT*, anthocyanidin 3-*O*-glucosyltransferase; (**K**–**O**) *MYBA1*, *MYBA2*, *MYB5a*, *MYB5b*, *MYBPA1*, transcription factor encode genes, belonging to the R2R3 Myb family, which controls the last steps in the anthocyanins biosynthesis pathway.

Leucoanthocyanidin dioxygenase (*LDOX)* and *UFGT* successively catalyzed the oxidation of colorless proanthocyanidins to form colored delphinidin or anthocyanins and the glycosylation of catalytically unstable anthocyanins to form various stable anthocyanins. The expression levels of the LDOX-encoding gene *VvLDOX*, the UFGT-encoding gene *VvUFGT*, and the regulatory genes *VvMYBA1* and *VvMYBA2* in the pericarps of the three red varieties were higher than those of the white varieties. Both the *VvUFGT* and *VvMYBA* genes were hardly expressed in the three white varieties during the véraison period (Figure 5H,J–L). The expressions of regulatory genes *VvMYBA5a* and *VvMYBPA1* in the 10 wpf grape berry skins of each cultivar were higher than at 11 wpf (Figure 5M,O).
