*2.7. Identification of Transcription Factors Regulating Petal Color Formation*

Transcription factors (TFs) are important regulators that play key roles in regulating the expression of the genes during anthocyanin biosynthesis in flowering plants [30]. According to the annotation in Plant TF Database v4.0, a total of 302 TFs from 36 families were identified in the *R. pulchrum*, of which 162, 139, and 179 TFs were found in the comparisons of D vs. A, C vs. A, and B vs. A (Figure 7a and Table S5). Most of the genes encoding TFs were down-regulated in the A sample relative to the B, C, and D samples. In the D vs. A comparison, 101 TFs were up-regulated and 61TFs were down-regulated. In the C vs. A comparison, 95 TFs were up-regulated and 44 TFs were down-regulated. In the B vs. A comparison, 96 TFs were up-regulated and 83 TFs were down-regulated. Moreover, 11 TF families containing more than 10 DEGs were identified: ERF (42), MYB (29), *bHLH* (24), WRKY (20), MYB related (16), FAR1 (15), NAC (14), LBD (12), C2H2 (12), bZIP (12), and TCP (11) (Figure 7b). Notably, the ERF, MYB, *bHLH*, and WRKY families were relatively large TF families in the three pairwise groups (Table S5).

**Figure 7.** Differentially expressed TFs in the three pairwise comparisons. (**a**) Distribution of the TF families that include more than 10 DEGs. (**b**) Venn diagram of differentially expressed TFs in D vs. A, C vs. A, B vs. A comparisons.

Furthermore, we investigated ERF, MYB, and *bHLH* TFs to enhance our understanding of their involvement in regulating color formation in *R*. *pulchrum*. The overlaps among the three pairwise comparisons were calculated, and 56 TFs were found in the overlapping regions (Table S5). In our present study, five DEGs (*Rhsim02G0040100*, *Rhsim03G0176600*, *Rhsim07G0072500*, *Rhsim08G0214100*, *Rhsim12G0192100*) encoding ERF and three DEGs (*Rhsim03G0180500*, *Rhsim06G0163100*, *Rhsim09G0042000*) encoding MYB were up-regulated in the D vs. A, C vs. A, and B vs. A comparisons. Moreover, the expression level of one gene (*Rhsim08G0230000*) linked to *bHLH* was up-regulated in the A sample, as compared to the B, C, and D samples (Table S5).
