###### Abstract  
_Rchmannia glutinosa_, a member of the Scrophulariaceae family, has been widely used in traditional Chinese medicine since ancient times. The main bioactive component of _R. glutinosa_ is catalpol. However, the biogenesis of catalpol, especially its downstream pathway, remains unclear. To identify candidate genes involved in the biosynthesis of catalpol, transcriptomes were constructed from _R. glutinosa_ using the young leaves of three cultivars, Beijing No. 3, Huafeng, and Jin No. 9, as well as the tuberous roots and adventitious roots of the Jin No. 9 cultivar. As a result, 71,142 unigenes with functional annotations were generated. A comparative analysis of the _R. glutinosa_ transcriptomes identified over 200 unigenes of 13 enzymes potentially involved in the downstream steps of catalpol formation, including 9 genes encoding UGTs, 13 for aldehyde dehydrogenase, 70 for oxidoreductase, 44 for CYP450s, 22 for dehydratasees, 30 for decarboxylclasses, 19 for hydroxyles, and 10 for epoxidases. Moreover, two novel genes encoding geranial synthase (RgGES), which is the first committed enzyme in catalpol production, were cloned from _R. glutinosa_. The purified recombinant proteins of RgGES effectively converted GPP to geraniol. This study is the first to discover putative genes coding the tailoring enzymes mentioned above in catalpol biosynthesis, and functionally characterize the enzyme-coding gene in this pathway in _R. glutinosa_. The results enrich genetic resources for engineering the biosynthetic pathway of catalpol and iridoids. transcriptome; _Rchmannia glutinosa_; catalpol; biosynthesis; geraniol synthase +
Footnote †: journal: _R. glutinosa_  
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Footnote 1: email: l.s.j. iridoids of the 8\(\beta\)-series, such as seco-iridoids and their derivatives, while _route II_ gives rise to the compounds of 8\(\alpha\)-stereochemistry and _epi_-series, including catalpol, aucubin, and similar decarboxylated iridoid glucosides [8]. _Route I_ has been well studied through feeding and molecular experiments. It has been reported that _route I_ is initiated with the precursor geranyl diphosphate (GPP), which is generated by condensing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) from the mevalonate (MVA) and/or methylerythitol 4-phosphate (MEP) pathways. GPP is transferred to the monoterpene compound geraniol, then hydroxylated to produce 10-hydroxygeraniol and converted to 10-oxogeranial. The reduction and cyclization of 10-oxogeranial generate neepetalactol, and it is further modified by oxidation, glycosylation, hydroxylation, methylation, and a C-C bond cleavage reaction to produce series intermediates in sequence that are deoxylogenatic acid, deoxyloganic acid, loganic acid, loganin, and secologanin (Figure 1) [8]. Secologanin is considered the common precursor of seco-iridoids. The genes involved in this route were isolated from _Catharanthus roseus_ and several other plants (Figure 1) [9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19]. _Route II_ is identical to _route I_ until 10-oxogeranial is generated. From 10-oxogeranial, _route II_ splits into two branches, one of which produces 8-_epi_-deoxyloganic acid as follows: 10-oxogeranial-_epi_-nepetalactol-_8-epi_-deoxyloganic acid, which shares similar steps to _route I_, except for the different stereochemistry. The other branch involves 10-oxogeranial-_epi_-nepetalactol-_epi_-iridotrial-_b_-_b_-_b_-_b_-_b_-_epi_-deoxyloganic acid (Figure 1) [20; 21; 22]. Through a sequence of reactions, including hydroxylation, dehydration, decarboxylation, and epoxidation, 8-_epi_-deoxyloganic acid is modified to form catalpol via a series of intermediates, including mussanosidic acid, deoxygeniposidic acid, geniposidic acid, bartisoside, and aucubin (Figure 1) [23; 24].