# Diversity and Functional Evolution of Terpene Synthases in Rosaceae
In plants, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) are the precursors for monoterpenes, sesquiterpenes, and diterpenes, respectively; terpene synthase genes (TPSs) are responsible for converting them into a multitude of cyclic and acyclic terpenoids [12]. The characteristic catalytic function of TPSs is to generate multiple terpenoid products using one substrate, thus collectively contributing to numerous different structures of plant terpenoids in addition to modifying enzymes [12]. Generally, the TPS family is characterized by two large domains including the N-terminal domain (PF01397) and the C-terminal metal cofactor binding domain (PF03936) [14]. The N-terminal domain possesses a conserved RRX8W (R, arginine, W, tryptophan, and X, alternative amino acid) motif and the C-terminal domain contains two highly conserved aspartate-rich motifs, the DDxxD motif and the NSE/DTE motif. The DDxxD motif is involved in the coordination of divalent ion(s), water molecules, and the stabilization of the active site, and is found among all functional TPSs [3]. The NSE/DTE motif flanks the entrance of the active site and function in binding a trinuclear magnesium cluster [14; 15]. TPSs are split into seven subgroups based on their amino acid sequence relatedness, namely TPS a-g. The majority of TPSs in most plants fall into one or two clades and the TPS-d clade was only encoded in gymnosperms [15; 16]. Different TPS clades differ considerably in catalysate and sequence. As the largest clade, the TPS-a clade mainly encodes sesquiterpenes. TPS-b and TPS-g clades are clustered closely to TPS-a; the TPS-b clade contains the conserved R(R)X8W motif and usually encodes monoterpenes, while the TPS-g clade lacks the conserved R(R)X8W motif, and functions in producing mono- and sesquiterpenes [17]. The TPS-c clade is characterized by the \"DXDD\" motif but not the \"DDXXD\" that was detected in other clades, and mainly functions in producing diterpene products [17]. The TPS-d clade is only encoded in gymnosperms, and function in producing mono- and sesquiterpene products. The TPS-e/f clades are clustered closely, and mainly encode diterpene products [17]. TPS-a/b clades lack the N-terminal \(\gamma\) domain characteristic of diterpene synthases found in clades c, e, and f. In general, the biosynthesis of isoprene, monoterpenes, and diterpenes occurs in the plastid and the biosynthesis of sesquiterpenes occurs in the cytosol [17]. So far, the TPS gene family members have been characterized in many plant species. The sizes of TPS families in the majority of sequenced plants genomes range from 1 to 100. The TPS families probably evolved through duplication of genes followed by functional divergence [13]. The bryophyte _Physcomittella patens_ has a single TPS gene [13]. In _Arabidopsis thaliana_, _Vitis vinifera_, _Ocimum sanctum_, _Daucus carota_, tomato, and _Camellia sinensis_, 32, 69, 81, 19, 44, and 80 TPSs have been identified, respectively [13; 18; 19; 20]. However, not all of the TPSs were functional. In recent years, the genomes of Rosaceae species such as peach, plum, apple, pear, and strawberry have been sequenced, which has significantly promoted the related studies of this family. Although abundant terpen were characterized in different tissues of Rosaceae species [3; 4; 21], a comprehensive study on TPSs has not been reported in Rosaceae. In this study, we mainly focused on three subfamilies in Rosaceae that are of economic importance as food crops, and did not select the _Spirea_ subfamily, as the _Spirea_ subfamily is only composed of ornamental flowers; thus, we excluded it from this study. For each subfamily, we chose two representative species with available sequenced genomes of high quality (chromosome scale). Based on the annotated genomes, we carried out the identification, characterization, and metabolite pathway mapping predictions of all TPSs encoded by eight Rosaceae species, including three Prunoideae species, three Maloideae species, and two Rosoideae species.