# Diversity and Functional Evolution of Terpene Synthases in Rosaceae
## 2 Results
### Tissue-Specific Expression of TPSs in Rosaceae  
For the three representative Rosaceae species (_P. persica_, _M. domestica_, and _F. vesca_), based on RNA-seq data of different tissue development (ripe fruit, immature fruit, leaf), each tissue consisted of two replicates. We examined the tissue expression profiles of TPSs, as listed in Table S4, and observed that TPSs in the three Rosaceae species demonstrated tissue-specific expression (see Figures 6 and S10). There were about 31, 26, and 41 TPSs expressed in at least one tissue for _P. persica_, _M. domestica_, _F. vesca_, respectively; surprisingly, 11 of them were putative TPSs without both domains, such as _Fr.ves-TP561_, _Fr.ves-TP2_, _Pr.pLo-TPS42_, _Ma.dom-TPS42_, etc. Whereas many TPSs with both domains were not expressed in any of the three tissues, these findings indicated the complexity of TPSs expression. In ripe fruits, expressed TPSs were only detected in strawberries; for _M. domestica_ and _P. persica_, no TPSs were expressed in ripe fruits. In addition, we observed that most TPSs were exclusively or highly expressed in one tissue, as shown in Figure 7, such as _Pr.pLo-TPS13_, _Ma.dom-TPS28_, and _Fr.ves-TPS9_ genes; they were highly or merely expressed in immature fruit or leaf. We further examined the expression pattern between different TPS clades; for the three plants, different TPSs from TPS-a clade were widely distributed in different tissues, indicating the functional diversity of TPS-a clades. However, the expression from other TPS clades varied among species, such as TPS-b clade; its members in _M. domestica_, _Ma.dom-TPS28_, were only highly expressed in immature fruit, but its counterparts in _P. persica_, _Fr.ves-TPS11_ and _Fr.ves-TPS13_, displayed an opposite expression pattern despite their high homology. _Fr.ves-TPS11_ was highly expressed in leaf, while _Fr.ves-TPS13_ was highly expressed in immature fruit, indicating the function differentiation  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**TPSs** **C**ade & **TPS-a** & **TPS-b** & **TPS-g** & **TPS-c** & **TPS-e** & **TPS-f** \\ \hline \multicolumn{6}{c}{**a. monoTPS**} \\ \multicolumn{6}{c}{} \\ 1, (–) alpha-pinene & & & & **a.monoTPS** & & \\ 2, 5-linal & & & & 1, (–) alpha-pinene & & \\ synthase & **a. monoTPS** & & 1, (–) alpha-pinene & & & \\ **b. SesquiTPS** & 1, (–)-alpha-pinene & & 1, (–) alpha-pinene & & & \\ 1, (–) germercance D & synthase. C/P & & & 3, Tricyclene & & **a. monoTPS** & 2, 5-linal \\ synthase. C/P & 3, Tricyclene & & 2, S-linal & & & \\ 2, (38, 62)-resolution & synthase EROS, & & & **b. SesquiTPS** & & \\ synthase. P & C/P & & & 1, (–) germercance D & & **b. SesquiTPS** & \\ 3, (E. & **b. SesquiTPS** & & & & 2, (36, 62)-neolafter the recent duplication. Similarly, for TPS-g clade, its members (_Fr.ves-TPS9-12_) in _F. vesca_ and members (_Pr.pLo-TPS9, Pr.pLo-TPS9_) in _P. persica_ were only highly expressed in leaf, while the counterparts in _M. domestica_ (_Ma.dom-TPS1, Ma.dom-TPS15-18_) showed different tissue expression specificity. In addition, we found that the TPS-f clade showed a larger variation between three species. _F. vesca_ lost the TPS-f genes, _M. domestica_ had two copies that displayed a low expression level, while _P. persica_ owned three copies, which is higher than all other species. Two copies were relatively highly expressed in leaf, and one copy was expressed in immature fruit. They were predicted as s-linalool synthases; their product s-linalool is assumed to be an important flavor component in _P. persica_[2]. On the whole, the above finding indicated that TPSs in Rosaceae also underwent extensive expression differentiation after the split of sister lineages and gene duplication. Figure 6: Expression pattern of expressed TPSs in the three Rosaceae species (_P. persica, M. domestica, F. vesca_). Heat mapping of TPSs gene expression in three Rosaceae species. The x-axis represents different samples (ripe fruit, immature fruit, and leaf), the y-axis represents TPSs. There are two replicates for each tissue.