## 3 Results
### Functional Analysis of Petal-Expressed RcTPSs in Butterfly Rose  
Phylogenetic analysis was performed using the maximum likelihood method, including nine petal-expressed _RcTPSs_, two bud-expressed _RcTPSs_, and other characterized TPS genes (Figure 7). Of these, five _RcTPS-a_ genes expressed in MU petals were grouped into two clusters. _RcTPS39_ was clustered with some genes that can catalyze monoterreponid products, such as _FvPINS_ (strawberry) [45], _PcTPS2_ and _PcTPS5_ (_P. campanulate_) [47], _PdTPS1_ (_Prunus dulcis_) [48], and _MdPIN/CAM_ (_Malus domestic_) [49]. An evolutionary analysis on the TPS-a genes of Poaceae exhibited that some TPS-a members can convert  
Figure 6: Differential metabolites and genes involved in middle and downstream pathway of volatile terpene biosynthesis in different samples. (a) Heatmap of volatile terpenes in D3 and D1 samples. Clustering_distance_rows ‘euclidean’, clustering_method ‘complete’. (b) Volcano plot of differential volatile terpenes between D1 and D3 samples. (c) Volcano plot of differential terpene-related genes between D1 and D3 samples. (d) Emission abundance of geraniol and expression levels of three _RcTPS_ in D1 and D3 samples. _RhLIB2_ was used as an internal control. Data are presented as the mean \(\pm\) standard error (\(n=3\)). Asterisks indicate significant differences between D1 and D3 samples at **_p_ < 0.01; ***_p_ < 0.001 by Student’s _t_-test. GPP into monoterepenes derived from an initial C6-C1 closure [50]. Further studies will be conducted to investigate the ability of _RcTPS39_ to catalyze monoterepene products. The other four _RcTPS-a_ genes were mainly clustered with _TPSs_ that catalyzed (_E,E_)-FPP to C15 products by an initial C10-C1 or C11-C1 closure. _RcTPS32_, _RcTPS33_, and _RcTPS46_ might have the ability to convert (_E,E_)-FPP to produce sesquiterpenes. The four _RcTPS-b_ genes were grouped into three clusters. _RcTPS8_ and _RcTPS9_ were clustered with ocimene synthases, whereas _RcLINS_ was clustered with other linalool synthases. _RcTPS18_ was clustered together with other angiosperm \(\alpha\)-farresene synthases (AFSs) from _Malus domestica_ (_MAAFS_) [51], _Prunus campanulate_ (_PcTPS_) [47], peach (_PpTPS_) [52], _Glycine max_ (_GmAFS_) [53], _Populus trichocarpa_ (_PITPS2_) [54], tomato (_SITPS2_) [13], grapevine (_VvGwbOciF_) [55], and _Ricius communis_ (_RcSeTPS_) [56], forming an \(\alpha\)-farresene synthase cluster (Figure 7). All genes in this cluster were predicted or identified as being localized in the cytoplasm. Sequence analysis showed that _RcTPS18_ exhibited 59% to 66% identity with other AFSs of Rosaceae plants. _RcTPS18_ and other AFSs exhibited conserved structural features including the RRx8W motif, DDxxD motif, NSD/DTE motif, and H-\(\alpha\)1 loop. The last motif demonstrated function in the binding of the metal ion K\({}^{+}\) in _MAAFS_ (Figure 8a) [57]. Figure 7: Maximum likelihood phylogeny of petal-expressed _RcTPSs_ and other characterized terpene synthases. _RcTPSs_ are highlighted in blue. The sequences used in this analysis are listed in Table S5. The subfamilies are illustrated with different colors: TPS-a (purple), TPS-b (green), TPS-e/f (orange), and TPS-g (blue). The distribution of _RcTPS18_, _RcLIN-NERS1_, and _RcLIN-NERS3_ on the chromosome is very close, forming a gene cluster (Figure 3). Interspecific micro-synteny analysis found that the chromosome distributions and functions of these three TPS gene were relatively conserved in peach and strawberry (Figure 8b). _RcLIN-NERS1_ and its three collinear genes (_FalNES1_, _FalNES2_, and _PpTPS3_) showed same catalytic functions [21; 45; 52]. The _RcTPS18_ and _PpTPS2_ were colinear gene pairs, indicating that they may have similar functions. In order to verify the correlation between the expression level of _RcTPS18_ and emission amounts of (_E,E_)-\(\alpha\)-farmesene, volatile analysis and qRT-PCR were performed among D1, D3 and petals of _Rosa_ 'Qinglian Xueshi'.