*2.7. Transcriptome Analysis of OfXTH Genes under Different Ambient Temperature Treatment*

From our previous investigated results, ambient temperature of 19 ◦C significantly promoted the flower-opening process in *O. fragrans*, while ambient temperature of 23 ◦C inhibited the flowering process [15]. In our study, the flower buds were also treated under 19 ◦C and 23 ◦C conditions. In the 19 ◦C treatment for 2 and 5 days, the traits of flower buds were reached at S1 and S2 stage, respectively. However, the flower buds still remained in the S1 stage when treated at 23 ◦C for either 2 days or 5 days. Therefore, the flower buds were collected at the second and fifth day under 19 ◦C and 23 ◦C treatment for transcriptome sequencing, and then we explored which *OfXTHs* are involved in the regulation of flower-opening processes responding to ambient temperature changes. Based on log2(ratio of abundance) ≤−1 or ≥1 with FDR < 0.001, a total of 5702 and 8082 differentially expressed genes (DEGs) were identified in 2 d, 19 ◦C vs. 2 d, 23 ◦C and 5 d, 19 ◦C vs. 5 d, 23 ◦C, respectively (Table S2). Among these obtained DEGs, five and sixteen differentially expressed *OfXTH* genes were identified at the second and fifth day under 19 ◦C treatment relative to the control 23 ◦C, respectively (Table 3). The differentially expressed *OfXTH* genes including *OfXTH24*, *27*, *32*, *35*, and *36* were all induced by 19 ◦C at the second day. Compared with the control of 23 ◦C at the fifth day, fifteen upregulated *OfXTH* genes (*OfXTH4*–*7*, *12*, *14*, *23*–*25*, *27*, *28*, *31*–*33*, *35* and *36*) were identified under 19 ◦C treatment. The expression of five *OfXTH* genes, including *OfXTH24*, *27*, *32*, *35*, and *36,* were significantly increased at the second and fifth day. Then, qRT-PCR was employed to validate the differentially expressed *OfXTH* genes. Consistent with the transcriptome profile, the expression of *OfXTH24*, *27*, *32*, *35* and *36* were strongly induced by 19 ◦C (Figure 7).


**Table 3.** DEGs of *OfXTHs* treated at 19 ◦C and 23 ◦C for 2 and 5 days.

**Figure 6.** Expression profiles of *OfXTHs* at different flowering stages. Significant differences are identified by SPSS (version 22) with Duncan's test (*p* < 0.05) and are represented by different letters above the error bars. (**A**) *OfXTH* genes highly expressed in the S0 period. (**B**) *OfXTH* genes upregulated in the S1 and S2 period. (**C**) *OfXTH* genes upregulated in the S4 and S5 period.

**Figure 7.** The qRT-PCR analysis of five differentially expressed *OfXTH* genes treated at 19 ◦C and 23 ◦C. Significant differences are identified by SPSS (version 22) with Duncan's test (*p* < 0.05) and are represented by different letters above the error bars.

#### **3. Discussion**
