## 3 Discussion  
### _O. officinal_  
is widely grown in subtropical and temperate regions and used as a health food, but also has a high ornamental and medicinal value [22; 30]. Epiphytic or lithophytic herbs commonly suffer from adverse environmental conditions such as chilling, drought, and water deficit [22; 23; 29; 30; 31; 32]. Plant volatile terpen play critical roles not only in the formation of orchid floral stents, but also in response to environmental stresses [1; 2; 5; 6; 7]. TPS is the primary enzyme responsible for catalyzing the formation of monoterenes (C\({}_{10}\)), sesquiterpenes (C\({}_{15}\)), or diterpenes (C\({}_{20}\)) from the substrates GPP, FPP, or GGPP, respectively (Figure 1). Therefore, studies on floral stents have mainly focused on the identification and analysis of _TPS_ genes responsible for the biosynthesis of terpen [7; 24]. Herein, 34 _DoTPS_ genes were identified in the _D. officinal_ genome according to the conserved C-terminal and \(N\)-terminal domain of TPS, followed by manual verification (Table 1; Figure 3). TPS subfamilies belong to a medium-sized family, with various gene numbers (approximately 20-150) among different plant species [1; 10]. For example, 32 _AttPS_ genes were functionally discovered in _A. thaliana_[11]. A total of 14 _SmTPS_, 33 _OsTPS_, and 152 _VvTPS_ genes were found in _S. moellendorffii_, _O. sativa_, and _V. vinifera_, respectively [1; 10; 17; 18]. Furthermore, _TPS_ occupied 0.26 genes/M in the _A. thaliana_ genome (125 M) [33], 0.13 genes/M in _S. moellendorffii_ (106 M) [34], 0.08 genes/M in the _O. sativa_ genome (389 M) [35], 0.31 genes/M in _V. vinifera_ (487 Mb) [36], and 0.02 genes/M in _D. officinal_ (1.35 G) [22; 23; 24]. It is possible that tandem duplication may have occurred during evolution of the _D. officinal_ genome, mainly in the TPS-a and TPS-b subfamilies. Phylogenetic analysis showed that DoTPS proteins fall into four known angiosperm TPS subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f), with the exception of the gymnosperm-specific TPS-d and _S. moellendorffii_-specific TPS-h (Figure 3). TPS-b was the largest subfamily among the DoTPS proteins, followed by TPS-a, which was consistent with _Daucus carota_[14], but in contrast to other species such as _A. thaliana_ (22 of the 32 _TPS_ genes were _TPS-a_ genes) [12] and _S. Lycopersicum_ (12 of the 29 _TPS_ genes were _TPS-a_ genes) [17]. As illustrated in Table S12, 16 DoTPS proteins were annotated as monoterepense synthase, Fourteen DoTPS proteins were annotated as sesquiterpene synthase, and four DoTPS proteins were annotated as diterpene synthase. These four putative diterpene synthases harbored one TPS-c and three TPS-e/f proteins. TPS-e/f proteins can produce mono-, sesqui-, and di-terpenes [1]. All 14 sesquiterpene synthases and 16 monoterepense synthases were assigned to TPS-a and TPS-b, respectively (Figure 4; Table S12). For mono- and sesquiterpenes, subcellular location and availability of substrate are more important for the characterization of typical products produced in vivo. RhTPS6 was localized in the cytosol, and was deemed to be associated with the formation of a sesquiterpene (nerolidol) and several monoterepenes (myrcene, limonene, _cis_-ocimene, _trans_-ocimene, and terpinolene), suggesting that GPP and FPP might move from plastids to the cytosol [21]. TPS-a can produce monoterepenes in vitro, while TPS-b can produce hemi-, mono-, and sesquiterpenes in vitro [1]. TPS-a is an angiosperm-specific clade that is responsible for sesquiterpene or diterpene synthases, and can be further divided into monocotyledonous- and dicotyledonous-specific subgroups [1; 9; 10]. In _A. thaliana_, four _TPS-a_ genes encode cytosolic sesquiterpene synthases, while the other nine _TPS-a_ genes harbored transit peptides presumably encoding diterpene synthases, although their functions have not yet been fully investigated [12]. In _S. Lycopersicum_, all _TPS-a_ genes only encode sesquiterpene synthases, and 11 of 12 are cytosolic and not chloroplastic [17].