*3.7. Correlation Analysis of MYB-Related Regulatory Genes and Anthocyanin Synthesis Structure*

*VvMYB5a* and *VvMYB5b* were not significantly associated with structural genes in the anthocyanin synthesis pathway, which may not be directly involved in regulating the synthesis of anthocyanins. *VvMYBPA1* showed significant correlations with *VvCHS*, *VvF3 H*, *VvF3H*, *VvFLS*, and *VvLAR*, which may directly regulate the flavonoid pathway, anthocyanin synthesis, flavonol synthesis, and catechol synthesis in the anthocyanin synthesis pathway. *VvMYBA1* was positively correlated with *VvF3 5 H, VvLDOX*, and *VvUFGT*, which may be directly involved in regulating the synthesis of anthocyanins and regulating the *UFGT* catalytic formation of stable anthocyanin pathways. *VvMYBA2* was not significantly associated with structural genes in the anthocyanin synthesis pathway (Table 3, Figure 7). Among these, the regulation of *VvMYBA2* and *VvMYB5a* was not clear, while synthetic genes regulated by *VvMYBPA1* and *VvMYBA1* were clearly known.

**Table 3.** MYB-related regulatory genes related to anthocyanin synthesis structural genes. \*\* represents *p* < 0.01.


**Figure 7.** The relationships between the expression color scale of anthocyanin synthesis structural genes and regulatory genes in grape peels and the regulation of the *MYBA* gene. The blue dotted arrows represent the structural genes in the anthocyanin synthesis pathway regulated by the regulatory gene; the '?' indicates that the regulatory mechanism of the regulatory gene is not yet clear; the green-to-red color scale means the TPM values showed an increasing trend.

#### *3.8. Screening of Genes Involved in the Regulation of Metal Ion Binding*

When screening by sorting all gene expression levels (TPM values) in the three white grape varieties, a gene located on chromosome 19 (gene ID: Vitvi19g01871) was found to show the highest expression level. Its expression level was much higher than those of other genes, and the gene was highly expressed (almost the maximum) in the three red varieties. Interestingly, the expression levels of this gene in the pericarps of the three white the three red varieties during the same period were also different (Figure 8). From the comparison of 10 wpf and 11 wpf, this gene was upregulated at 11 wpf compared with 10 wpf in the white cultivar 'Italia', while the opposite was found in 'Muscat of Alexandria' and 'Rosario Bianco' (Figure 8). In the red varieties of 'Benitaka' and 'Rosario Rosso', the expression levels at 11 wpf were downregulated compared with 10 wpf, while the expression in 'Flame Muscat' was higher (Figure 8). According to the functional annotation, it was inferred that this gene encodes a metallothionein-like protein, which regulates the binding of copper ions and zinc ions. Copper ions are related to the synthesis of chlorophyll, which may have a certain impact on the change in peel color at the véraison stage.

**Figure 8.** (**A**) 'Italia' 'Benitaka' TPM value almost top 10 of gene expression 10 wpf; (**B**) 'Italia' 'Benitaka' TPM value almost top 10 of gene expression 11 wpf; (**C**) 'Muscat of Alexandria' 'Flame Muscat' TPM value almost top 10 of gene expression 10 wpf; (**D**) 'Muscat of Alexandria' 'Flame Muscat' TPM value almost top 10 of gene expression 11 wpf; (**E**) 'Rosario Bianco' 'Rosario Rosso' TPM value almost top 10 of gene expression 10 wpf; (**F**) 'Rosario Bianco' 'Rosario Rosso' Top TPM value almost top 10 of gene expression 11 wpf. The green line represents the TPM values of white cultivars; the black line represents the TPM values of red and black cultivar.

#### **4. Discussion**

In recent years, it has been observed that many important fruit varieties are selected by bud sport [41,42]. According to statistics, there have been thousands of bud sport types on fruit trees, and some fruit trees can form a variety of bud sports. Due to the particularity of each bud sport, it brings certain characteristics in germplasm resources during fruit production and breeding. Therefore, this is an important approach used in the breeding of fruit crops.

The present study revealed that *VvMYBA1* showed elevated expression levels in the three red sport varieties at 10 wpf compared to three white varieties. In addition, after 11 wpf there were significantly higher *VvMYBA1* gene expression levels compared with the white cultivar grapes (Figure 4B). The *VvMYBA1* gene was proved to be a key transcription factor regulating color change in grape berry skins [43]. The *VvMYBA1* gene was expressed only in red berries, while it was hardly expressed in white berries (Figure 5K).

According to a correlation analysis, the majority of genes or enzymes related to the anthocyanin synthesis pathway were significantly correlated with *VvMYBPA1* and *VvMYBA1* (Table 3). Among them, five genes showed significant correlations with various genes, such as *VvMYBPA1*, *VvCHS*, *VvF3 H*, *VvF3H*, *VvFLS*, and *VvLAR*. Significant correlations of *VvF3 5 H*, *VvLDOX*, and *VvUFGT* with *VvMYBA1* were observed in our study.

The expression of the flavonoid 3-*O*-glucosyltransferase (UFGT) gene is essential for anthocyanin biosynthesis in grapes [44]. The *VvMYBA1* gene normally regulates the expression of *VvUFGT*, a key upstream gene of anthocyanin synthesis [45] considered to be the last step for catalyzing anthocyanin synthesis in the anthocyanin biosynthesis pathway [46], and both are very important in the formation of grape skin color. The RNA-Seq results indicated that the expression trends of *VvUFGT* in the three red varieties were consistent; among them, the expression level in 'Benitaka' was significantly higher than in the other two varieties and was not expressed in white grape varieties (Figure 5J). The above results are consistent with the results of a previous study conducted on 'Italia', 'Benitaka' and 'Flame Muscat' [5]. The Pearson's correlation analysis showed that *VvMYBA1* and *VvUFGT* were highly correlated with the same expression trend (Figure 5J,K). The results also indicated that *VvMYBA1* positively regulated the *VvUFGT* gene and played an important role in the biosynthesis of anthocyanins.

According to previous reports on anthocyanin synthesis in apples and bilberries, it was found that *MYBPA1* could also regulate the expression of *UFGT* [47]. In this experiment, the correlation between these two genes was not high. This may explain why, among the three groups of varieties (the 'Italia' vs. 'Benitaka' group, the 'Muscat of Alexandria' vs. 'Flame Muscat' group, and the 'Rosario Bianco' vs. 'Rosario Rosso' group), *MYBPA1* was not a key transcription factor regulating *UFGT* and the anthocyanin biosynthesis pathway. Its specific regulatory mechanism still needs further study.

*MYBPA1* plays an important role in the anthocyanin biosynthesis pathway, and the expression of *MYBPA1* is positively correlated with anthocyanin accumulation [48]. In blue bilberries, the *MYBPA1* and *MYBA* transcription factors can activate the expression of DFR and ANS genes in the anthocyanin biosynthesis pathway, which are considered key genes for anthocyanin biosynthesis [49]. In this study, the expression levels of the *VvMYBPA1* gene in the two groups of bud sport varieties of 'Italia' vs. 'Benitaka' and 'Muscat of Alexandria' vs. 'Flame Muscat' were higher at 10 wpf compared with 11 wpf, while this gene was not expressed in the 'Rosario Bianco' vs. 'Rosario Rosso' group (Figure 5O). The expression trends of five structural genes (*VvCHS*, *VvF3'H*, *VvF3H*, *VvFLS*, and *VvLAR*, Table 3) related to *VvMYBPA1* were different in the three groups of tested varieties (Figure 5A–E,I). The phylogenetic analysis depicted that the flavonoid-related R2R3 MYBs of *VmMYBPA1* and *VvMYBPA1* belonged to the same group. *VmMYBPA1* could regulate the expression of *CHS* and significantly regulated the expression of the *F3 5 H* gene, while *VmMYBPA1* expression was significantly decreased in white mutant berries compared with blueberries [50], which indicates that it was related to anthocyanin biosynthesis. The expression level of *MYBPA1* is associated with the accumulation of proanthocyanidins (PA) during the early development

of grape berries. The expression level of *MYBPA1* was lower before the véraison stage and peaked at two weeks following the véraison stage, later showing a low expression level. *MYBPA1* activates the promoters of *LAR* and *ANR* in grapes [51]. The expression of *VvMYBPA1* was opposite to that of *VmMYBPA1*, as expressed in 'Italia'. This is in contrast to previous studies showing no expression observed in white grape varieties. A previous study also found that *VvMYBPA1* could also be expressed in seeds [52]. The above results might indicate that the pathway or regulation mechanism of the *MYB* gene in anthocyanin synthesis is different in diverse species.

The gene expression analysis showed that the expression of *VvLDOX* was consistent with the expression trends of *VvMYBA1* and *VvUFGT* in other test materials, except for in 'Italia' (Figure 5H,J–K). *LDOX* has a unique expression pattern in the biosynthesis of anthocyanin in grape peels, and its expression levels were very high in red or black peels, which was related to the content of anthocyanin. *UFGT* is present in many tissues of grape, as well as in the skins of white and red grape varieties, while the expression of *LDOX* is not as absolute as *UFGT* [53]. *VvMYBPA1* was found to activate *VvLDOX* expression in grapes [49], and this result suggested that the expression of *VvLDOX* in 'Italia' may be related to the regulation of *VvMYBPA1*, while there was no significant correlation between *VvMYBPA1* and *VvLDOX*.

In addition to the above results, an interesting point found in this study was the gene located on chromosome number 19 (Gene ID: Vitvi19g01871). The gene expression levels (TPM values) of green varieties at 10 weeks and 11 weeks post-flowering were between 64,711–168,489 and 78,173–127,381, respectively. The expression levels of red grape varieties at 10 weeks and 11 weeks post-flowering were between 39,130–59,249 and 28,319–67,849, respectively. The expression levels of this gene in green varieties were much higher than those in red varieties, as well as much higher than all the other differentially expressed genes (Figure 8). The gene was annotated by GO molecular gene function as a metallothionein-like protein that regulates the binding of copper ions to zinc ions. Copper ions play an important role in the redox of plant respiration and are related to chlorophyll synthesis, which is important for photosynthesis. Increased photosynthesis and chlorophyll lead to excessive chlorophyll accumulation in grape peel cells. However, its mechanism of action in the process of bud sport peel color and anthocyanin synthesis is still unclear, and the function of this gene needs to be further verified at molecular or cellular levels.
