*4.3. Phylogenetic Tree Construction and TPS Classification*

The full-length sequences of TPSs protein sequence with both domains from Rosaceae species were used to perform sequence alignment and phylogenetic tree construction using MEGA7 program [34]. The ClustalW method was used for sequence alignment under default parameters, the Maximum Likelihood and Neighbor-joining methods based on the Jones-Taylor-Thornton (JTT) matrix-based model were used for phylogenetic tree construction, a Bootstrap method was used for the phylogeny test with 500 replications, rates among sites was Gamma distributed (G), and Partial deletion was used for gaps/missing data treatment, with site coverage cutoff at 90%. The produced tree was further embellished by the FigTree program (http://tree.bio.ed.ac.uk/, accessed on 2 September 2021). TPS members were classified by their clustering relation with the query sequence mentioned above. For each TPS clade, their TPS member's sequences were further aligned by ClustalW method, and a sequence logo for the visualization of the highly conserved aspartate-rich motif in C-terminal domain was produced by TBTools [33].
