## 3 Results and Discussion
### Transcriptome Analysis of Grapes Receiving Different Cluster-Thinning Treatments  
Three grape treatments were collected at three developmental stages: when berries began to color and enlarge (E-L 35), had intermediate Brix values (E-L 36), and were harvest-ripe (E-L 38) to obtain nine cDNA libraries, with three replicates (27 total libraries), named H-EL35, H-EL36, H-EL38, M-EL35, M-EL36, M-EL38, L-EL35, L-EL36, and L-EL38 (Table S3). After removing ambiguous nucleotides, low-quality reads and adapters, each library contained 19,446,318\(\sim\)26,848,251 clean reads. Of the total clean reads, 88.09% to 92.40% were uniquely matched to the grape reference genome ([[http://plants.ensembl.org/Vis_vinifera/Info/Index](http://plants.ensembl.org/Vis_vinifera/Info/Index)]([http://plants.ensembl.org/Vis_vinifera/Info/Index](http://plants.ensembl.org/Vis_vinifera/Info/Index)), 26 August 2007). The average Q30 and Q20 values were 92.77 % and 97.35 %, respectively. The average mapped reads ranged from 38,688,187.67 in L-EL38 to 42,127,556.67 in M-EL36. Hierarchical clustering and a PCA of the 27 samples based on FPKM suggested that our sequencing data were reliable (Figure 2a,b). Hierarchical clustering showed that the global gene expression varied distinctly from one berry developmental stage to another, but the variations between cluster thinning treatments were not significant (Figure 2a). The PCA map showed that PC1 and PC2 accounted for 78.50% and 15.60% of the total variance, respectively (Figure 2b). Similar to the hierarchical clustering, there was significant separation among the three developmental stages, implying that the expression levels of many genes were altered during berry development. To identify the DEGs related to cluster thinning, we compared the FPKM values of L to H, L to M and M to H at three fruit ripening stages. At E-L 35, 235 DEGs were obtained in L vs. H, among which 94 were upregulated and 141 were downregulated (Figure 3a). There were 897 DEGs in L vs. M, among which 802 were upregulated and 95 were downregulated (Figure 3b). In M vs. H, there were 537 DEGs, among which 52 were upregulated and 485 were downregulated (Figure 3c). Then, the DEGs identified in L vs. H, L vs. M and M vs. H were compared to pinpoint those common to the three treatments. In total, 10 genes were differentially and constitutively expressed among the three treatments (Figure 3d). At E-L 36, 732 DEGs were detected in L vs. H, among which 617 were upregulated and 115 were downregulated (Figure 4a). There were 211 DEGs in L vs. M, among which 120 were upregulated and 91 were downregulated (Figure 4b). In M vs. H, there were 603 DEGs, among which 85 were upregulated and 518 were downregulated (Figure 4c). There were 49 common DEGs differentially and constitutively expressed in L vs. H, L vs. M and M vs. H (Figure 4d). There were 925 DEGs in L vs. H at E-L 38, among which 307 were downregulated and 618 were upregulated (Figure 5a). In L-EL 38 vs. M-EL 38, there were 172 DEGs including 67 downregulated and 105 upregulated genes (Figure 5b). In M-EL 38 vs. H-EL 38, there were 435 DEGs, among which 302 were upregulated and 133 were downregulated (Figure 5c). Only six DEGs were common to the three treatments (Figure 5d). Figure 2: Heatmap clustering showing correlation among samples based on global expression profiles (**a**) and principal component analysis of the samples based on gene expression profiles (**b**). Figure 3: Differential expression volcano map of L vs. H, L vs. M, and M vs. H at E-L 35 stage, respectively (**a–c**). Venn diagram depicting the shared and unique differentially expressed genes (DEGs) identified by RNA-Seq analysis between grapes at the E-L 35 stage (**d**). Each point in the differential expression volcano map represents a gene, and the abscissa represents the logarithm of the multiple of the differential expression of a gene in the two samples. The ordinate represents the negative logarithm of statistically significant changes in gene expression.