*2.7. 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-TPS61*, *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

after 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. The rows and columns were clustered based on row-scale normalized expression values. TPSs clades were shown on the right by different colors; putative TPSs without both domains are indicated by black stars.

**Figure 7.** The expression level of TPSs from clades b, c, g, e, and f in the three Rosaceae species (*P. persica*, *M. domestica*, *F. vesca*). Tissues (immature fruit, leaf) are indicated by different colors.
