## 3 Results
### Identification of Putative Genes in the Pathway of Catalogl Biosynthesis
The members of Family 1 PSPGs are responsible for the glycosylation of various natural plant products [10]. The phylogenetic analysis of RgUGT1-9 showed that RgUGT1,2,3,9 fell into group G, while RgUGT4 and RgUGT6 belonged to group O and F, respectively. RgUGT1 and RgUGT9 were closely related to CrUGT8 (Figure 6B, Supplementary Table S8). Furthermore, the qRT-PCR analysis revealed that RgUGT1 and RgUGT9 were highly expressed in the young leaves compared to the roots (Figure 6C). Figure 6: **(A)** The heatmap showing an expression clustering of putative UGT genes in catalpol biosynthesis; **(B)** phylogenetic analysis of RgUGT1-9 with other known UGTs from different groups. The tree was constructed with the Neighbor-Joining method (with 1000 bootstrap replications) in the MEGA 7.0 software. Accession numbers of UGTs were shown in Supplementary Table S8; **(C)** the qRT-PCR analysis of UGT candidate genes in catalpol formation. Asterisks indicate a significant difference (*** \(p\) < 0.001). It has been predicted that ALDs/NADP\({}^{+}\) oxidoreductaseases might catalyze the oxidation of boschnaloside to form 8-_epi_-deoxyloganic acid. In the transcriptomic databases, 104 genes for aldehyde dehydrogenagenases and 439 genes for NADP\({}^{+}\) oxidoreductase were identified. Among them, 13 aldehyde dehydrogenase genes and 70 NADP\({}^{+}\) oxidoreductaseense genes met the gene screening criteria and showed significantly higher expression levels in the young leaves than those in the tuberous and adventitious roots (Supplementary Tables S9 and 10). The cytochrome P450 enzymes (CYP450s) exhibit a broad range of activities in hydroxylation, epoxidation, decarboxylation, as well as C-C oxidative cleavage reactions. CYP450s might participate in a series of modifications in catalpol biosynthesis (Figure 1) [38]. A total of 363 unigenes were annotated as CYP450s while the expression level of 44 genes correlated well with the catalypol content in _R. glutinosa_ (Supplementary Table S11). Given that the reported CYP450 genes, including G10H, DLS, D1/H, and SLS in _route I_ of iridoid biosynthesis, belong to the CYP72 and CYP76 families, these 44 candidate sequences were further screened, resulting in 12 CYP72 and 7 CYP76 sequences, respectively (Supplementary Table S11). Among these CYP450 members, Cluster-11149.22060 showed the highest sequence similarity with the reported acubin synthase [26], which exactly met the gene screening criteria of this study (Supplementary Table S11). The dehydration of musaaensodic acid may be catalyzed by dehydatase. In the _R. glutinosa_ transcriptomes, there were 85 sequences annotated as dehydatase, of which 22 candidates showed higher expression levels in the young leaves compared with the roots, and met the screening criteria (Supplementary Table S12). The transformation of geniposidic acid to bartsioside can take place by decarboxylation, which is usually catalyzed by decarboxylase. By keyword searching, a total of 239 decarboxylase unigenes were found, and the expression level of 30 sequences matched the accumulation pattern of catalpol (Supplementary Table S13). Hydroxylase and epoxidase also participate in the biosynthesis of catalpol. Hydroxylase is proposed to catalyze the hydroxylation of 8-_epi_-deoxyloganic acid, deoxygeniposidic acid, and bartsioside, while epoxidase can convert aucubin to form catalpol. A total of 19 genes for hydroxylases and 10 genes for epoxidases, which met the gene screening criteria herein, were predicted to be involved in catalpol formation in _R. glutinosa_ (Supplementary Table S14). Among these hydroxylase candidates, Cluster-11149.14301 and Cluster-11149.10607 were annotated as iridoid hydroxylase and exhibited higher expression levels in the young leaves than in the roots (Figure 7). The heatmap of all the candidate genes in the biosynthetic pathway of catalpol is shown in Supplementary Figure S5.