## 4 Discussion
glutinosa_ cultivars (Beijing No. 3, Huaifeng, and Jin No. 9 cultivars) to elucidate the catalpol biosynthetic pathway in this study. Moreover, considering the difference in the catalpol content among different tissues, the transcriptomic data from the tuberous and adventitious roots were further obtained. The transcriptomes yielded a total of 71,142 unigenes with functional annotation. The number of functionally annotated unigenes was more than that in the tuberous root transcriptome from the _R. glutinosa_ Wen 85-5 cultivar, but less than that generated from root databases from 1706, BJ1, Wen 85-5, and QH1 cultivars [27; 45]. This difference might result from the use of different cultivars and tissues in different studies. The transcriptome from the adventitious roots which had no catalpol was first applied to reduce the number of candidate genes in catalpol biosynthesis. The adventitious root library can provide more reliable evidence for the comparative transcriptome analysis. According to the HPLC detection results, the rationale for discovering genes in the catalpol biosynthetic pathway was designed as follows: first, compared with the adventitious roots, candidate genes would show higher expression in the young leaves with fold changes larger than two, and second, the transcript level of the selected genes would be highest in the young leaves, followed by the tuberous roots and the adventitious roots, in sequence. In addition, the candidate genes would be present in all the Beijing No. 3, Huaifeng, and Jin No. 9 cultivars. Using the comparative transcriptome analysis, more candidate genes in the downstream pathway of catalpol biosynthesis were obtained in this study. As a typical iridoid glucoside with 8\(\alpha\)-stereochemistry, catalpol may be formed by _route II_: geraniol\(\rightarrow\)10-hydroxygeraniol\(\rightarrow\)10-oxogenarial\(\rightarrow\)_epi_-nepetalactol\(\rightarrow\)_8-epi_-deoxylogane acid or boschnaloside\(\rightarrow\)_8-epi_-deoxyloganic acid. 8-_epi_-deoxyloganic acid is subsequently modified to catalpol by hydroxylation, dehydration, decarboxylation, and epoxidation (Figure 1) [8; 22; 24]. However, the knowledge of genes responsible for the biosynthetic steps in _route II_ is poor. In this study, the formation of catalpol is divided into two periods to conduct analysis. The first period is the production of 8-_epi_-deoxyloganic acid from GPP, which shares similar steps with _route I_. The first step of this period is the conversion of GPP to geraniol by GES. A GES candidate gene (Cluster-11149.40193) was found in _R. glutinosa_, and it showed 71% amino acid identity with the GES from _C. roseus_. It is predicted that the transformation of geraniol to 8-_epi_-deoxyloganic acid is catalyzed by common enzymes, such as G10H, 10 HGO, _epi_-ISY, and DLS-like. Using keyword and blast searching approaches, all these common enzymes were found here. Nine sequences encoding these four enzymes met the gene screening criteria in this study. These candidates showed identical expression features to the accumulation pattern of catalpol (Figure 5), indicating their possible roles in catalpol production. Glycosylation reactions in secondary metabolism are usually catalyzed by glycosyltransferases from Family 1 PSPSGs [10]. A total of 167 UGT sequences were identified, and the transcript level of eight unigenes (RgUGT1-8) matched the catalpolaccumulation. However, the other UTG sequence Cluster-11149.23607 (RgUGT9) showed the highest amino acid identity with CrUGTS, a reported UGT in iridoid biosynthesis. The phylogenetic analysis suggested that RgUGGT1,2,3,9 belonged to group G (Figure 6), which contains several iridoid-specific glucosyltransferasearase members, indicating these unigenes might play roles in the catalpol biosynthesis in _R. glutinosa_. Considering that sequence homology is not enough to predict the precise enzymatic activity of glycosyltransferase, RgUGT1-3 as well as RgUGT9 will be further cloned to analyze their functions.