## 2 Results
### AsA Synthesis Pathway Analysis in Kiwifruit  
Based on existing studies of AsA anabolism in _Arabidopsis_ and kiwifruit [10], orthologous genes in the L-galactose pathway were identified using BlastP with a \(p\) value \(<\) 1 \(\times\) 10\({}^{-5}\). Enzymes of the L-galactose pathway analyzed in the transcriptome included all orthologues of D-fructose-6-P (_PMI_), D-mannose-6-P (_PMM_), D-mannose-1-P (_GMP_), GDP-D-mannose (_GME_), GDP-L-galactose (_GGP_), L-galactose-1-P (_GPP_), L-galactose (_GDH_), and L-galactone-1,4-lactone (_GLDH_). As shown in Figure 4A, Actinidia36324 of _PMI_, Actinidia36057 and Actinidia6047 of _GMP_, and Actinidia09998 of _PMM_ were hardly expressed. Actinidia26306, Actinidia34962, Actinidia19079, and Actinidia02420 were slightly expressed, and their expression decreased as the developmental stages of kiwifruit progressed, whereas the expression of Actinidia36370 and Actinidia02420 showed an increasing trend. Actinidia40102 and Actinidia04499 of _GME_ and Actinidia05074 and Actinidia32270 of _GGP_ were highly expressed and had similar expression patterns. In _A. lufidia_, all of these genes reached the highest expression at DAP40. In _A. rufu_, their expression was downregulated with the progression of fruit developmental stages. Notably, the expression of Actinidia32270 was the highest among all genes in the L-galactose pathway, and its expression was similar to the trend of AsA synthesis in kiwifruit (Figure 4A). Furthermore,  
Figure 2: Differently expressed genes (DEGs) between _A. lufidia_ and _A. rufu_ during different developmental stages. (**A**) Intraspecific pairwise comparison of differentially expressed genes at different developmental stages. (**B**) Interspecific comparison of differentially expressed genes at the same developmental stage. (**C**) Venn diagram of DEGs between different kiwifruit species at the same developmental stage. (**D**) Gene ontology (GO) analysis of significantly expressed genes in (**C**), summarizing biological processes, cellular components, and molecular functions.  
we performed RT-qPCR to confirm the expression level of the genes of the L-galactose pathway in transcriptome analysis. We find there is a high correlation (average \(r>65\%\)) between the RT-qPCR and RNA-seq for those genes (average FPKM \(>1\)). Especially, the candidate gene \(GGP3\) (Actinidia32270) had the highest expression, and its expression pattern was highly consistent with transcriptome analysis. Therefore, our results provide an overview of transcripts encoding the enzymes involved in AsA biosynthesis and clear clues for associated studies.  
We predicted cis-regulated elements in the promoter regions of structural genes in the L-galactose pathway. As shown in Figure S3A, most of the cis-acting elements play roles in responding to the abiotic environment or plant growth and development. Based on the 'HongYang v3' genome as a reference, we further predicted 2264 transcription factors, mainly including the bHLH, MYB, and C2H2 transcription factor families (Figure S3B). Our results identified candidate gene pools for AsA anabolism in kiwifruit.  
Figure 3: The filtered DEGs whose expression is consistent with the change of AsA concentration in _A. latifolia_ and _A. rufu_. The line plot is showing DEGs expression patterns (**A**) in four different developmental stages and the bar plot is showing the GO enrichment results of these DEGs (**B**) in _A. latifolia_. The line plot shows DEGs expression patterns (**C**) in four different developmental stages, and the bar plot shows the GO enrichment results of these DEGs (**D**) in _A. rufu_.