## 2 Results
### Identification of 33 Unigenes Involved in Terpenoid Backbone Biosynthesis  
Based on the integrated annotation process, 71 unigenes were annotated as genes involved in terpenoid backbone biosynthesis (Table S4), which showed that similarity-based BLASTX searching using Swiss-Prot database was a more powerful approach than other methods. After further manual annotation by searching the NCBI non-redundant protein sequence database using BLASTX method, we found 27 unigenes with high similarity to genes in bacteria or animals, and those genes were all expressed in root or flower with very low expression levels (Table S4), which may be caused by contamination of microorganisms in those organs. Two unigenes (TRINITY_DN82648_c0_g1 and TRINITY_DN82648_c0_g3) were annotated as different parts of one gene. Six unigenes shorter than 500 bp, together with four unigenes did not match the criteria for functional annotation. Thus, we found 33 unigenes that were annotated as terpenoid backbone biosynthesis genes in _S. barbata_, 32 of which had full length cDNA  
Figure 2: Length distribution of assembled genes. Genes with lengths larger than 5 kb are counted together. The dashed line represents average length of all unigenes. We obtained nearly all known terpenoid backbone genes compared to _Sa. miliorrhiza_ (Figure 3A; Table S5). _Sa. miliorrhiza_ is a Lamiaceae species with available genome sequences [35], thus giving us an opportunity to investigate the species-specific evolution of these genes. Some gene expansions were observed in these two species and they have the same gene families except for MDD and HMGR. In _S. barbata_, MDD form a small gene family that contains two members compared with the single gene in _Sa. miliorrhiza_, while HMGR was encoded by a smaller gene family containing only three genes in _S. barbata_ compared with the four genes in _Sa. miliorrhiza_. Genes in _S. barbata_ have similarities with genes in _Sa. miliorrhiza_ from 79.9% to 97%. Figure 3: Schematics of the putative terpenoid biosynthetic pathway and the expression level of identified genes from _S. barbata_. (**A**) the putative terpenoid biosynthetic pathway in _S. barbata_. Numbers in the brackets indicate the gene family numbers of each enzyme. (**B**) a clustered heat map for the 33 terpenoid backbone genes and 14 TPS genes with log-2 transformed FPKM value in root (RT), flower (FL), and aerial parts (AP). Purple marked enzymes are involved in the MVA pathway, and blue marked enzymes are involved in the MEP pathway. The newly identified enzyme IPK in alternative MVA pathway is highlighted in purple. Abbreviations: AACT, aceto-acetyl-CoA thiolase; CMK, 4-(cytidine 5\({}^{\prime}\)-diphospho)-2-C-methyl-d-erythitol kinase; DXS, 1-deoxy-p-xylulose 5-phosphate synthase; DXR, 1-deoxy-d-xylulose-5-phosphate reductioisomerase; FPS, Farnesyl pyrophosphate synthase; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; GGPPS, Geranylgeranyl pyrophosphate synthase; MCT, 2-C-methyl-d-erythnitol 4-phosphate cytidyltransferase; IDI, Isopentenyl diphosphate isomerase; IPK, Isopentenyl phosphate kinase; MDS,2-C-methyl-d-erythnitol 2,4-cyclodextrin diphosphate synthase; MDD, Mevalonate diphosphate decarboxylase; MDS, 2-C-methyl-d-erythnitol 2,4-cyclodextrin diphosphate synthase; MVK, mevalonate kinase; Phos, phosphatase(s); PMK, phosphomevalonate kinase; TPS, terpene synthases (including monoterpene synthases); CPS, copalyl diphosphate synthase; KSL, Kaurene synthase. (**A**) shows (**B**) shows. In order to uncover possible biological functions and the relationship between these genes, hierarchical clustering based on the log-2 transformed FPKM value in root, flower, and aerial parts was performed. The genes were divided into three distinct groups (Figure 3B). Six genes, HMGR2 in the MVA pathway, DXS3 in the DXP pathway, and IDI2, GGPPS2, GPPS.LSU2, and GPPS.SSUI were included in group I.