## 2 Results and Discussion
### Candidate Genes for Terpene Synthases  
Within our assembled contigs, 17 unique terpene synthase sequences were identified (Figure 2). Of these sequences, two could be identified as being involved in general metabolism, with contig 30,041 annotated as ent-kaurene synthase and contig 8677 as a cycloartenol synthase. The contigs 43,795 and 24,682 were not assigned putative functions based on the phylogenetic tree in Figure 2 due to incomplete sequences; however, they appear to group with triterpene synthases of general metabolism. The remaining 13 were located within clades corresponding to genes involved in the specialized metabolism of mono- and sesquiterpenoid biosynthesis. Of these, only contig 820 could be assigned a putative function because of its close homology to the recently described kunzeaol synthase from _T. garganica_[14]. To determine a definitive function, the remaining 12 contigs would need to be cloned, expressed and biochemically characterized in order to determine their terpene products. Nevertheless, phylogenetic analysis indicates that contigs 7414, 36274, 18983, and 509 are likely to produce sesquiterpene backbones, while contigs 13346, 16049, 25105, 25300, 29053, 31363, 33553, and 43413 are all likely to be involved in monoterenoid biosynthesis (Figure 2). The number of terpene synthases identified here, 8 monoterpene synthases and 5 sesquiterpene synthases, is slightly larger than the number found in _Arabidopsis_, where 4 mono and 3 sesquiterpene synthases were identified [33], while _Artemisia annua_ contains at least 4 mono and 6 sesquiterpene synthases (based on a NCBI search of published sequences). This indicates that the number found in _Thapsia_ corresponds well to what can be expected of mono and sesquiterpene synthases in one plant species. The relatively large number of terpene synthases found in _T. laciniata_ roots is in contrast to _T. garganica_ roots, where only two sesquiterpene synthases were found in the transcriptome in a recent study [14]. This is in agreement with the larger chemical diversity of complex terpen found in the roots of _T. laciniata_, which is in accordance with several studies on the chemistry of _Thapsia_ plants [5,6]. The sesquiterpene synthases described here are currently undergoing biochemical characterization. In order to confirm that the assembled contigs generated using the Trinity assembler were accurate, we designed primers to anneal around the predicted start and stop codons of contigs 820, 7414 and 509, representing three predicted full-length terpene synthases with expected lengths of approximately 1600 bp. All three genes could be readily amplified from cDNA synthesized from RNA isolated from a different _T. laciniata_ plant than was originally sequenced (Figure 3). Subsequent sequencing of these nucleotides demonstrated that the sequences were exactly as predicted by the contig assembly, demonstrating that our contig collection is likely to be an accurate representation of nucleotide sequences. The three genes amplified are currently undergoing biochemical characterization. **Figure 2.** Phylogeny tree of terpene synthases. The tree illustrates the likely enzymatic function of 17 _T. laciniata_ contigs. The figure clearly shows that comp820 is likely to be a Kunzeaol synthase, which is part of the guaianolide biosynthesis. The tree also shows a distribution of the _Thapsia laciniata_ sequences throughout the three main clades of the tree.