*2.2. Phylogenetic Analysis of HSF Genes in M. sativa*

We created a NJ phylogenetic tree using MEGA6.0 for the amino acid sequences of 16 *M. sativa* HSF, 21 *A. thaliana* HSF, and 25 *O. sativa* HSF in order to study the evolutionary relationships of *MsHSF* genes (Figure 1). Based on the well-established *A. thaliana* family classification, HSF are clearly classified into three groups, namely HSF A (green), HSF B (yellow) and HSF C (blue). Members of MsHSFs were identified in all three groups. The largest was MsHSF A, which made up 52.2% of the overall MsHSF, which was broken down into 9 subgroups (A1–A9). HSF A consists of nine proteins, namely MsHSF01, MsHSF04, MsHSF05, MsHSF07, MsHSF09, MsHSF10, MsHSF11, MsHSF12 and MsHSF16. MsHSF proteins were not aggregated into the three subgroups, A4, A7 and A9. MsHSF B was divided into 5 subgroups (B1–B5), accounting for 39.1%, and consisted of 6 members (MsHSF02, MsHSF03, MsHSF06, MsHSF08, MsHSF13, and MsHSF15). MsHSF C was the smallest group. Also, it contained only one MsHSF14. *M. sativa* HSF proteins clustered more closely with *A.thaliana* HSF proteins, according to the phylogenetic tree, which is interesting. This finding shows that the HSF proteins of dicotyledons and monocotyledons have distinct evolutionary differences.

#### *2.3. Multiple Sequence Alignment and Protein Modeling Analysis of the HSF Gene in M. sativa*

To check for the presence and position of conserved protein structural domains, we used Jalview to perform a multiple sequence alignment on 16 MsHSF proteins. We found that the DBD structural domain, which contains roughly 100 amino acids, is substantially conserved among all members of the MsHSF family (Figure 2). The DBD structural domain has three helix bundles (1–3) and four reverse parallel folds, as predicted from protein secondary structure (1–4). Additionally, we predicted the protein 3D structure of HSF of *M. sativa* and labeled the starting position of the 1 DBD structural domain in the figure (Figure 3).
