# Identifying Terpenoid Biosynthesis Genes in _Euphoria maculata_ via Full-Length cDNA Sequencing
## 3 Results
### Terpenoid Biosynthesis Pathway Genes  
We identified all genes in the MVA, MEP, and terpenoid biosynthesis pathways (Table 5; Figure 7). The nucleotide sequences of paralogous genes and isoforms in these pathways are listed in Supplementary Table S1. In the MVA pathway, six genes encode the enzymes involved in IPP biosynthesis, with one (AAC thiolase and MVA kinase) to five (HMG-CoA reductase) paralogs per gene and one to three isoforms of each paralog. The first reaction in the MEP pathway is the condensation of pyruvate with glyceraldehyde 3-phosphate to form DOXP by DOXP synthase. The DOXP synthase gene had two paralogs and one and three isoforms of each paralog. There are five genes involved in the conversion of DOXP to 1-hydroxyl-2-methyl-2-(\(E\))-butenyl-4-diphosphate (HMBPP), which had one (CDP-ME synthase) to five (HMG-CoA reductase) paralogs and one to three isoforms of each paralog. HMBPP is reduced to dimethylallyl diphosphate or IPP by IPP/DMAPP synthase, which has two paralogous genes with only one isoform each. IPP and DMAPP are isomers that are interconverted by IDI. IDI has two paralogous genes with one and three isoforms. IPP undergoes head-to-tail dimerization to form geranyl diphosphate (GPP) by GPP synthase, which has two paralogous genes with a single isoform each. GPP is converted to monoterpenes by monoterpene synthase, which has two paralogous genes with a single isoform each. GPP is also converted to famesyl diphosphate (FPP) by farnesyl synthase, which is encoded by a single-copy gene with two isoforms. FPP is processed into sesquiterpenoids or squalene by sesquiterpene synthase or squalene synthase, respectively. Squalene is further processed to triterpenoid by triterpene synthase, which is encoded by three paralogous genes with a single isoform each. Geranylgeranyl diphosphate (GGPP) is converted into diterpenes by diterpene synthase, which is annotated as ent-kaurene synthase. Ent-kaurene synthase is encoded by a single-copy gene with one isoform. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Enzymes** & **Abbreviation** & **Pathway** & **No of Paralogs** & **Range of Isoform** \\ \hline \multicolumn{4}{c}{Acetate-Mexolonate} \\ Acetoacetyl CoA thiolase & AAC thiolase & 1 & 1 \\
3-Hydroxy-3-methylglutaryl synthase & HMG-CoA Syntase & 3 & 1 \\
3-Hydroxy-3-methylglutaryl reductase & HMG-CoA Redutase & 5 & 1–3 \\ Mevalonate kinase & MVA kinase & 1 & 1 \\ Mevalonate phosphate kinase & MVAP kinase & 2 & 1–2 \\ Mevalonate diphosphate decarboxylase & MVAP carboxylase & 2 & 1–2 \\ \multicolumn{4}{c}{Non-Mexolonate} \\
1-deoxy-D-xylulose-5-phosphate reductoisomerase & DOXP reductoisomerase & 3 & 1–3 \\ Cytidine diphosphate 2-C-methyl-D-erythnitol synthase & CDP-ME synthase & 2 & 1 \\ Cytidine diphosphate 2-C-methyl-D-erythnitol kinase & CDP-ME kinase & 1 & 1 \\
2C-methyl-D-erythnitol synthase & MECP synthase & 4 & 1 \\
1-hydroxy-2-methyl-2-D-butenyl4-diphosphate synthase & HMBPP synthase & 2 & 2 \\ IPP/MDAPP synthase & IspH & 2 & 1 \\ \multicolumn{4}{c}{Terpenoid synthesis} \\ Isopentenyl-diphosphate delta-isomerase & IDI & 2 & 1–2 \\ Geranyl diphosphate synthase & GPP synthase & 2 & 1 \\ Farnesyl diphosphate synthase & FPP synthase & 1 & 2 \\ Geranyl geranyl diphosphate synthase & GGPP synthase & 2 & 1 \\ Monoterpene synthase & Monoterpene synthase & 2 & 1 \\ Sesquiiterpene synthase & Sesquiiterpene synthase & 1 & 1 \\ Diterpene synthase & Ent-Kaurene synthase & 1 & 1 \\ Squalene synthase & Squalene synthase & 2 & 1 \\ Triterpene synthase & Triterpene synthase & 3 & 1 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Enzymes involved in the biosynthesis of terpenoids, isopentyl diphosphate, and dimethylallyl diphosphate. In a single gene, different paralogs had different numbers of isoforms as exemplified by the DOXP gene in Figure 3. DOXP:para1 had three isoforms with different termination sites, and DOXP:para3 had two isoforms with different starting and termination sites, as well as different exons. The expression of the paralogs differed among the tissues (Figure 7).