## 2 Results
### Differential Expression Analysis  
As previous studies demonstrated that differentially expressed genes play important roles in the accumulation of nutrients in plant fruits [26], differential expression analysis was carried out in intraspecific pairwise comparisons between fruit samples at different developmental stages in _A. latifolia_ and _A. rufa_. A total of 28,648 nonredundant genes were identified as differentially expressed genes (DEGs) in intraspecific pairwise comparisons (Figures 2A and S1A). Most DEGs (11,132) were found in the comparison between DAF40 and DAF120 in _A. latifolia_ and in the comparison between DAF60 and DAF120 in _A. rufa_ (Figure 2A). Furthermore, we found that the number of DEGs between adjacent developmental stages was less than that between interval stages in each of these kiwifruit species (Figures 2A and S1A), suggesting that the differential expression of genes gradually increased during fruit growth. Except for the comparison between DAF60 and DAF120 in _A. rufa_, the number of downregulated DEGs was higher than that of upregulated DEGs in the different intraspecific pairwise comparisons (Figure 2A), indicating that the gene expression levels of most DEGs decreased with the development of fruit in both _A. latifolia_ and _A. rufa_. This also suggested a low transcriptional activity of more DEGs in more mature plant tissues. To further explore the regulation of species divergence in kiwifruit AsA synthesis, DEGs between _A. latifolia_ and _A. rufa_ at the same developmental stage were also identified. We found that the average number (9181.66) of DEGs in intraspecific pairwise comparisons was lower than that (13,545.75) in interspecific comparisons (Figures 2A,B and S1A,B), suggesting that gene expression levels had more differences between different kiwifruit species than between intraspecific stages during fruit development. Our results showed the most DEGs between these two species at DAF60, which was probably because the difference in AsA content was the largest at DAF60 among the four stages (Figures 1B and 2A,B).  
In addition, we further investigated conserved and stage-specific DEGs between _A. latifolia_ and _A. rufa_ using a Venn diagram (Figure 2C). As expected, the most stage-specific DEGs (3988) were found in the comparison of DAF60 fruit between these two kiwifruit species, while the fewest stage-specific DEGs (1685) were found in the comparison at DAF40. This further suggested that many biological processes dramatically changed from DAF40 to DAF60 in kiwifruit. We also identified 3925 conserved DEGs in comparisons of _A. latifolia_ and _A. rufa_ at the four stages (Figure 2C). Further GO enrichment analysis of these conserved DEGs suggested that most DEGs were enriched in response regulation pathways, such as'response to biotic stimulus','response to external stimulus','response to stress', and'response to chemical' (Figure 2D). We screened genes that were differentially expressed in each of the comparisons between adjacent developmental stages for both _A. latifolia_ and _A. rufa_. The expression patterns of these DEGs in the two species were similar to the AsA content of the fruits at the four stages (Figures 1B and 3A,C). Furthermore, GO enrichment analysis showed that these DEGs were significantly enriched in'responses to various abiotic stimulus', specifically enriched in'response to light stimulus' in _A. latifolia_ (Figure 3B); whereas the DEGs were enriched in'secondary metabolic processes' (Figure 3D) in _A. rufa_.  
Therefore, we speculated that the changes of external environmental factors probably play important roles in AsA synthesis.