## 3 Discussion  
Ascorbic acid (AsA), which is an essential nutrient to maintain growth and health in humans and animals, acts as an antioxidant that efficiently reacts with and detoxifies a number of reactive oxygen species (ROS), participates in plant respiration and photosynthesis, and maintains the normal metabolic functions of plants [28; 29; 30]. Fresh fruits and vegetables are the main sources of AsA for humans, and a high AsA concentration is a significant feature of kiwifruit. However, the AsA levels of different kiwifruit species vary greatly [18], and the molecular metabolic mechanism of AsA in kiwifruit is still unclear. In this study, we investigated the patterns of AsA synthesis in _A. tufolia_ (AL) and _A. tufa_ (AR) at four developmental stages by transcriptome analysis. Approximately 1.16 billion high-quality reads were generated. Genome data for _A. tufolia_ and _A. tufa_ are not yet available; therefore, we characterized the transcriptomes of AL and AR using Illumina-based RNA sequencing with the 'HongYang v3' genome as a reference, and an average of 66.68% of the reads were uniquely aligned against the reference genome (Table S1). In addition, three orthologues of \(GGP\), which is a candidate gene involved in AsA synthesis, were verified by RT-qPCR, and the expression patterns of \(GGP\) were similar to those of RNA-seq analysis (Figure 4), demonstrating the reliability of our transcriptome data. Moreover, all samples had high biological reproducibility, indicating the high accuracy of our sampling and sequencing (Figure 1C). The accumulation of AsA in kiwifruit is a dynamic process during growth and development. Our results showed that the accumulation patterns of AsA in two kiwifruit species involved rapid synthesis in the early developmental stages and a gradual decline in the later stages (Figure 1B), which is consistent with previous studies [31; 32]. Differential expression analysis showed that there were large numbers of differentially expressed genes between different kiwifruit species at the same developmental stage (Figures 2A,B and S1B),  
Figure 9: Single nucleotide polymorphisms (SNPs) analysis. (**A**) Venn diagram presenting SNPs analyzed in _A. tufolia_ and _A. tufa_. (**B**) Distribution of SNPs in _A. tufolia_ and _A. tufa_ on the ‘HongYang v3’ genome chromosome, in which _A. tufolia_ is red and _A. tufa_ is green. (**C**) Distribution of SNPs in _A. tufolia_ and _A. tufa_ on _Actinidia_32270 of chromosome 10 in the ‘HongYang v3’ genome. The red box represents a conserved domain PLN03103 of _Actinidia_33270. which we attribute to changes in the expression patterns of _A. latifolia_ and _A. rufa_ genes during fruit development. A Venn diagram comparison of these differentially expressed genes showed that 3925 DEGs were significantly differentially expressed between _A. latifolia_ and _A. rufa_ (Figure 2C), and the distribution of SNPs on the genomic chromosomes was also found to be specific between _A. latifolia_ and _A. rufa_ (Figure 9). We speculate that these DEGs and the specificity of the SNPs may largely determine the large difference in AsA content between _A. latifolia_ and _A. rufa_. Further screening and GO enrichment analysis of these DEGs indicated that they functioned mainly in response to various abiotic signals, such as response to light (Figure 3B). We hypothesized that changes in photoperiod or light quality have a regulatory effect on AsA synthesis in kiwifruit. However, earlier studies in tomatoes and _Arabidopsis_ demonstrated that AsA anabolism is correlated with light intensity [33], photosynthesis [34], photoperiod [35], and photoinhibition [36]. In a cis-regulated element prediction of promoters of structural genes in the L-galactose pathway [37], it was found that most elements were mainly responsive to the abiotic environment, including light responsiveness (Figure S3A), which further supported our conjecture.