## 2 Results
### Weighted Gene Co-Expression Network  
To explore the stage correlations of the candidate genes in the AsA synthesis pathway, the datasets of the _A. latifolia_ and _A. rufa_ expression matrices across different stages (DAF20, DAF40, DAF60, and DAF120) were subjected to WGCNA. After removing silenced and constantly expressed genes, a total of 16,876 genes were used to build weighted gene co-expression networks. Based on linkage hierarchical clustering coupled with topological overlap dissimilarity and a dynamic tree-cut measure, the filtered genes were assigned to 11 co-expression modules designated with random colors (Figures 5A and S4A and Table S3). Eight of these modules were significantly (\(p\) value \(<\) 0.05) relevant to one of the four developmental stages of fruit, among which the turquoise and blue modules were significantly correlated with DAF20, the dark turquoise and gray modules were significantly correlated with DAF40, the red module was significantly correlated with DAF60, and the magenta, dark-green, and dark-gray modules were significantly correlated with DAF120 (Figures 5A and S5 and Table S3).  
Figure 4: Expression patterns of expressed genes in the L-galactose pathway assigned to AsA synthesis in the DAF20, DAF40, DAF60, and DAF120 transcriptomes of _A. latifolia_ (AL) and _A. rufa_ (AR). (**A**) Log-transformed expression values range from \(-\)9 to 12, and pink and blue indicate up- and downregulated transcripts, respectively. (**B–G**) The expression levels of three homologous genes of _GGP_ (Actinidia05074, Actinidia36370, and Actinidia32270) of _A. latifolia_ and _A. rufa_ at different developmental stages. Error bars indicate \(\pm\) SD (\(n\) = 3).  
We further analyzed the percentage of transcription factors in the hub genes in different modules. Hub genes were defined as genes that were significantly (correlation coefficient > 0.8) associated with one module, further indicating that these genes were strongly associated with a specific stage (Figure S4B). Our results suggested that transcription factors were only significantly enriched in the gray module that was significantly related to DAF40 (chi-square test), suggesting that more transcription factors in the gray module are associated with the period at DAF40 (Figure S4B). Combined with the dramatic increase in AsA content at DAF40 in _A. latifolia_, we speculated that transcription factors in the gray module might contribute to the high AsA content in _A. latifolia_. In addition, we constructed several secondary networks of structural genes of the L-galactose pathway, showing the co-expression relationships between structural genes in the AsA synthesis pathway (red),  
Figure 5: Co-expression network during kiwifruit development. (**A**) Network hubs regulating genes in the differentiation stages. Colors indicate genes displaying peak expression in the corresponding stages. (**B**) Constructed secondary network of structural genes in the L-galactose pathway. Structural genes are shown in red, transcription factors are shown in green, and other genes with co-expression relationships are shown in blue.  
transcription factors (green), and other genes (blue) (Figure 5B and Table S3). The results suggested that these seven structural genes play a more important regulatory role in the AsA synthesis pathway (L-galactose pathway) than other structural genes, and their co-expressed transcription factors might also be involved in the regulation of AsA synthesis.  
Taken together, our transcriptome analysis identified one orthologue of _GGP3_ (Actinidia33270) in the L-galactose pathway showing high expression in fruit and a close correlation with the AsA content (Figures 1B and 4) as an important candidate regulator of AsA synthesis in kiwifruit (Figure 5B). Therefore, we selected Actinidia33270 to carry out a transgenic assay in kiwifruit calli and further verified the effect of _GGP3_ on AsA synthesis.