## 3 Results  
### Metabolomics Analysis of Three Raspberry Cultivars Leaves  
A non-targeted UPLC-MS/MS-based metabolomics technique was used to discover the differentially accumulated metabolites of three raspberry cultivars leaves. In the leaves of the three kinds, according to the VIP value (VIP \(>\) 1) and _t_-test (_p_ \(<\) 0.05) screening conditions, a total of 399 metabolites with significant differences were found (Supplement Table S1). Figure 1A,B show PCA analysis of detected metabolites in different cultivar leaves and quality control (QC) samples. QC samples were clustered closely in the PCA diagram of metabolites, indicating that the experimental stability and repeatability are reliable. The values of Autumn Bliss and Autumn Britten are focused in the negative half of the _Y_-axis, whereas the values of Red Autumn are concentrated in the positive half. PCA analysis is an unsupervised analysis method that struggles to explain the relationship between the contents of the principal components. OPLS-DA analysis was utilized to further evaluate the metabolic data in order to better discern the differences between samples. The metabolites of Autumn Bliss, Red Autumn, and Autumn Britten leaves were clustered in three different groups by OPLS-DA, showing that the three cultivars' metabolites profiles were significantly different (Figure 1C,D). The OPLS-DA was validated using 10-fold cross validation, and the evaluation parameters Q2 (predictive power) and R2 (fitting power)values were both larger than 0.5. (Figure 1E,F). Differential metabolites might then be examined and filtered based on the variable importance in the projection (VIP) values greater than one. Figure 1: Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) of metabolite accumulation levels in three raspberry cultivars. Three cultivars groups are shown in their names, respectively. The X and Y axes represent the first principal component (PC1) and the second principal component (PC2) in positive ion mode (**A**), negative ion mode (**B**) for PCA, positive ion mode (**C**), negative ion mode (**D**) for OPLS-DA, 10-fold cross validation of negative ion mode (**E**) for OPLS-DA, 10-fold cross validation of positive ion mode (**F**) for OPLS-DA, respectively. Differential accumulated metabolites in three cultivars were compared using multi-dimensional and one-dimensional analysis. The VIP value was utilized to determine the impact of each metabolite on group discrimination. Autumn Britten vs. Autumn Bliss (Table 1) had 36 up-regulated metabolites and 65 down-regulated metabolites; Autumn Britten vs. Red Autumn (Table 2) had 48 differentially up-regulated metabolites and 108 down-regulated metabolites; Autumn Bliss vs. Red Autumn (Table 3) had 88 differentially up-regulated metabolites and 54 down-regulated metabolites, indicating that leaf metabolites were significantly different. The significance of difference accumulated metabolites between groups (VIP \(>\) 1, \(p\) < 0.05) was verified using a _t_-test, which was represented by volcano plots between groups, as shown in Figure 2. The metabolites differentially accumulated among the groups were indicated by pink dots in the figures, and the average accumulation levels were calculated using log2 (fold change) values.