## 3 Results
### Functional Annotation and Classification of the R. glutinosa Unigenes  
A total of 71,142 unigenes were successfully annotated by searching all these sequences against seven public databases, including the Nr, Nt, Pfam, Swiss-Prot, KEGG, KOG, and GO. Among these databases, the highest annotation percentage was in the Nr (38,788 unigenes; 54.52%), followed by the Nt database (30,861 unigenes; 43.37%) and the Swiss-Prot (29,731 unigenes; 41.79%) (Table 2). \begin{table}
\begin{tabular}{c c c c c c c c} \hline
**Sample** & **Raw Reads** & **Clean Reads** & **Clean Bases** & **Error Rate** & **Q20 (\%)** & **Q30 (\%)** & **GC (\%)** \\ \hline J9L & 31,180,906 & 29,195,338 & 8.76 G & 0.03 & 97.84 & 93.43 & 45.42 \\ J9R & 31,674,784 & 30,297,505 & 9.09 G & 0.03 & 97.85 & 93.38 & 44.44 \\ J9 AR & 30,827,652 & 29,371,270 & 8.81 G & 0.03 & 97.75 & 93.19 & 45.10 \\ BJL & 30,882,434 & 29,153,263 & 8.75 G & 0.03 & 97.83 & 93.36 & 45.02 \\ HFL & 31,220,770 & 29,902,124 & 8.97 G & 0.03 & 98.01 & 93.91 & 44.47 \\ \hline \end{tabular}
\end{table} 
Table 1: Statistics of sequencing of _R. glutinosa_ transcriptomes. Figure 2: **(A)**_R. glutinosa_ plant; **(B)** the content of catalpol in the young leaves of different _R. glutinosa_ cultivars. BJL, young leaves of Beijing No. 3; HFL, young leaves of Huaifeng; J9L, young leaves of Jin No. 9; **(C)** the concentration of catalpol in different parts of _R. glutinosa_. YL, young leaf; OL, old leaf; R, tuberous root. Error bars represent the standard errors of the means (SEMs) calculated from three biological replicates. Asterisks indicate a significant difference (_p_ < 0.05). The KOG annotation revealed that 10,119 unigenes were classified into 25 terms, of which the largest category was \"posttranslational modification, protein turnover, chaperones\", including 1367 unigenes, followed by \"general function prediction only\" (1232 unigenes) and \"translation, ribosomal structure, and biogenesis\" (1150 unigenes) (Figure 3A, Supplementary Table S3). To further explain the function of unigenes in biological pathways, all the unigenes were searched against the KEGG database. The result showed 13,862 unigenes were classified into 34 KEGG pathways (Figure 3B, Supplementary Table S4), of which \"signal transduction\" (1757 unigenes), \"carbohydrate metabolism\" (1350 unigenes), and \"translation\" (1340 unigenes) were the highest represented pathways. To functionally classify the _R. glutinosa_ transcriptome, 28,511 unigenes were categorized into three GO terms: biological process, cellular component, and molecular function. The subcategory for \"cellular process\" (16,395 unigenes) was the most abundant group, followed by \"metabolic process\" (15,485 unigenes) and \"binding\" (15,155 unigenes) (Figure 3C, Supplementary Table S5). The FPKM method was applied to calculate the transcript abundance of the unigenes. Given that the content of catalpol is highest in the young leaves, followed by the tuberous roots and adventitious roots, the DEGs between the different tissues of _R. glutinosa_ were screened. A total of 16,241 DGEs were obtained from the J9L library versus the J9 AR library, including 5421 up-regulated DEGs and 10,820 down-regulated DEGs (Figure 4A). The GO annotation of these up-regulated genes showed that the highest number of unigenes was under the term of \"catalytic activity\" (1662 unigenes), followed by \"single-organism metabolic process\" (886 unigenes). For the up-regulated genes in the J9L library, the top three enriched pathways were \"starch and sucrose metabolism\", \"carbon fixation in photosynthetic organisms\", and \"glyoxylate and dicarboxylate metabolism\" (Figure 4B). By comparing the J9L library with the J9R library, 5139 up-regulated DGEs as well as 3545 down-regulated DGEs were identified. The up-regulated genes were mainly classified into the pathway \"carbon fixation in photosynthetic organisms\" (Figure 4B).