## 3 Results  
### Changes of Metabolome in Pear during Development and Storage  
Significant differences in the metabolites of pears among different stages were detected. PCA is a common method to assess the omics context and reduce the dimensionality of large data sets. The results of PCA show that the fit degrees of different biological replicates in the same stage are good, indicating that the samples in this experiment are representative. Except for the similarity between the fruit development of Z1 and S stages, the fruit development was quite different between the Z2-5, S1-4 and Z1 stages. In the first principal component (PC1) with a variation interpretation of 74.63%, all samples were divided into two parts, indicating that in terms of metabolic substances, the contents of aroma substances in the fruits during the growth period and the later storage period were quite different. However, in the second principal component (PC2) with a variation interpretation of 15.39%, there was no significant difference among fruits at different stages (Figure 1A). Notably, the sum of the variation explanation of PC1 and PC2 reached 90.02%, indicating that the differences in all samples distributed in PC1 were the most reasonable. Therefore, the PCA results show that the contents of pear metabolites in each stage are stable and can provide a good theoretical support for the search of key metabolites. The number of DEMs in adjacent stages is an effective way to digitally measure the difference between two stages (Figure 1B). The total number of DEMs in all comparison groups was 172. In the comparison group of adjacent stages, the difference in development stages is stable, while the difference in storage stage fluctuates largely. The trend in the number of up-regulated differential metabolites is consistent with that of the total differential metabolites. Specifically, changes in the S2-Z1 process were relatively stable. The numbers of differential metabolites in the Z1_vs_Z2 and Z3_vs_Z4 groups were the largest and both increased significantly. The number of down-regulated differential metabolites decreased gradually in the development stage and remained stable in the storage stage from S1-Z5. In the S1_vs_S2 comparison group, 90% of the metabolites were down-regulated. In the Z1_vs_Z2 comparison group, the total number of differential metabolites and the number of up-regulated differential metabolites were both at the peak, but the number of down-regulated metabolites accounted for only 17.07%. These results indicate that fruit metabolism is active in the early stage of pear development. The metabolism of pear fruits changed the most at the early stage of storage, and then the changes tended to be stable. The main purpose of metabolomic analysis is to detect and screen out biologically and statistically significant metabolites from biological samples and thereby to clarify the metabolic process and change mechanism of organisms. According to the results of PCA and correlation analysis, S1, S2, S3, S4 and Z1 can be combined into group R, and Z2, Z3, Z4 and Z5 can be combined into group S (Figure 1A). Then, the two groups were analyzed independently. An Upset map can uniformly show unique or common differential metabolites between different groups (Figure 1C,D). In group R (Figure 1C),  
Figure 1: Differences in aroma components of pear fruits at different stages. (**A**) PCA of metabolomics. (**B**) Total distribution of differential expressed metabolites (DEMs) in different comparison groups. (**C,D**) Numbers of common and specific DEMs among different comparison groups. the most unique differential metabolites in the comparison group of the adjacent stages were found in S2_vs_S3 (57), and the least number of differential metabolites was detected in S4_vs_Z1 (46). The S2_vs_S3 group and the S1_vs_S2 group contained at most 16 and only 7 specific differential metabolites, respectively. Notably, the number of differential metabolites shared by the four comparison groups was only four.