## 2 Results
### Genes Related to Catalogl Biosynthesis in R. glutinosa  
Based on precursor feeding experiments, researchers established the pathway of catalpol biosynthesis and inferred that catalpol and other iridoid glycosides were generated from geraniol, which was produced through a combined biosynthetic route involving non-mevalonate (MEP) and mexolonate (MVA) pathways [19; 20]. [PERSON] et al. reported that the complete biosynthetic pathway of catalpol has been deciphered for all possible intermediates with their corresponding enzymes in _Picorhiza kuroa_ (Figure 6A) [21]. The putative genes encoding enzymes in the upstream of the iridoid pathway, including geraniol synthase (GES), geraniol 10-hydroxylase (G10H), cytochrome P450 reductase (CPR), and 10-hydroxygeraniol oxidoreductase(10 HGO), have been identified in _R. glutinosa_[16]. However, the genes encoding enzymes in the downstream of _R. glutinosa_ catalpol biosynthesis are still unknown. In this study, we identified 155 unigenes encoding 16 enzymes involved in the catalpol biosynthesis of _R. glutinosa_, including 102 unigenes encoding seven enzymes upstream of the iridoid pathway, including 1-deoxy-D-xylulose 5-phosphate reductio isomerase (DXR), geranyl diphosphate synthase (GPPS), isopentenyl-diphosphate Delta-isomerase (IPI), GES, G10H, CPR, and 10 HGO. Most interestingly, we also identified 53 unigenes encoding enzymes of the downstream of the catalpol biosynthesis pathway, including iridoid synthase (IRS), cytochrome P-450 monooxygenase (CPM), aldehyde dehydrogenase (ALD), flavanone 3-dioxygenase (F3D), uroporhyrinogen decarboxylase  
Figure 5: Top 20 enriched KEGG pathways among the annotated DETs across four comparisons. (**A**) Significant enrichment pathways for genes in 1706_RS vs. 1706_nRS comparison. (**B**) Significant enrichment pathways for genes in BJ1_RS vs. BJ1_nRS comparison. (**C**) Significant enrichment pathways for genes in QH1_RS vs. QH1_nRS comparison. (**D**) Significant enrichment pathways for genes in W85_RS vs. W85_nRS comparison. The \(X\)-axis below represents KEGG pathways, and the \(Y\)-axis indicates the enrichment factor. Low q-values are shown in blue, and high q-values are depicted in white. Point size indicates DET number (more: big, less: small). (UPD), UDP-glucuron acid decarboxylase (UGD), and squalene monooxygenase (SQM), in our _R. glutinosa_ transcriptome. Expression analysis of 155 genes involved in the catalpol biosynthesis pathway was done in radial striation and non-radial striation of _R. glutinosa_ tuberous root for the four cultivars. The results showed that 16, 11, 9, and 9 genes were upregulated in 1706, BJ1, QH1, and W85 non-radial striations compared to their radial striations, respectively (Table S8). Four unigenes, _CL1003.Contig2_ (GPPS),  
Figure 6: Expression patterns of catalpol biosynthetic unigenes between radial striations and non-radial striations of _R. glutinosa_. (**A**) Biosynthetic pathway of catalpol. (**B**) Heat map representing expression dynamics of unigenes involved in biosynthesis. The expression values (FPKM) for unigenes were \(\log_{10}\) transformed and scaled across each row, and the heatmap was generated by MultiExperiment Viewer (MeV). (**C**) Relative expression of candidate genes in involved in catalpol biosynthesis. These genes expression levels were all determined by Real-time PCR (qRT-PCR). Vertical bars indicate the standard deviation of three biological replicates. Asterisks (*) indicate a significant difference at the \(p\) < 0.05 level.