*2.4. Gene Structure and Conserved Motif Analysis of the HSF Gene in M. sativa*

HSF proteins were split into three categories, HSF A, HSF B, and HSF C, based on the phylogenetic analyses mentioned above (Figure 4A). Our analysis of the MsHSF gene structure revealed that *HSF* genes from the same group typically have a similar number of introns in their structures. There is just one intron and two exons shared by all *HSFB* and *HSFC* genes in *M. sativa* (Figure 4B). Except for *MsHSF12*, which had eight introns, the *M. sativa HSF A* genes ranged in intron count from one to four. Three genes included two introns; three genes (*MsHSF01, MsHSF07*) contained four introns; two genes (*MsHSF10, MsHSF11*) contained one intron. *MsHSF09* and *MsHSF016* contain four introns.

**Figure 1.** Model for phylogenetic analysis of HSF in *M. sativa*. Each subgroup is distinguished by a different color.

**Figure 2.** Conserved domain alignment of MsHSF members.

We also used MEME to identify up to 10 highly conserved motifs in each HSF protein (Figure 4C). The results show that the relative positions of most of the sequence motifs of the same group are similar. All MsHSF contain motifs 1 and 3, which constitute the most highly conserved part of the DBD, and the absence of motif 2 in some genes indicates that motif 2 is not necessary in the highly conserved part of the DBD. Motifs 7 and 9 were found only in HSF A, and motif 8 was present only in HSF B. These results suggest that the biological functions of the MsHSF proteins that are grouped together may be similar, and that different patterns may be related to different functions in separate subgroups.

**Figure 3.** Three-dimensional model of the protein of MsHSF family members, with the starting position of the α1 DBD structural domain indicated by 1. (**A**–**P**) in the figure represent the 16 proteins of the *M. sativa* HSF family, respectively.
