*2.3. Gene Structure and Motif Analyses*

Gene Structure Display Server (GSDS) was used to investigate the *OfXTHs* gene structure. The number of exons in thirty-seven *Of XTHs* ranged widely within 3–5 (Figure 2). Only *OfXTH23* contained two exons. The conservative motif, ExDxE, was present in every OfXTH protein. Motifs 3, 5, 8, and 9 were highly conserved in all proteins. Group I/II genes contained common motifs 2–9, while OfXTH16 lacked motifs 4, 6, 7, and OfXTH34 lacked motif 7. In addition, motif 10 was only present in proteins of Group I/II. Motifs 1–5 and 7–9 were found in Group IIIA, and motifs 1–3, 5, 8 and 9 presented in Group IIIB. In Ancestral Group, OfXTH28 shared the same motif with other OfXTHs, except for motif 2.

**Figure 2.** Gene structure and motif distribution of OfXTH proteins. (**A**) Gene structure, putative signal peptide sequences, and conservative motif sequence. Each subgroup is distinguished by a different color. White boxes and black lines represent exons and introns, respectively. The green and red strips indicate putative signal peptide sequences and conservative motif sequence, respectively. (**B**) The motif distribution. Motifs 1–10 in the OfXTH proteins are represented by different colored boxes.

#### *2.4. Chromosomal Location and Synteny Analysis*

As shown in Figure 3, 38 *OfXTHs* were distributed on the 17 chromosomes. Notably, seven genes, *OfXTH14*–*20*, were densely located on the Chr08, which was the chromosome distributing with the largest number of *OfXTH* genes. No *OfXTH* gene located on Chr05, Chr07, Chr14, Chr17, and Chr21. In addition, the other chromosomes contained 1–4 *OfXTH* genes. The locations of *OfXTH* genes on the chromosomes are described in detail in the mentary Materials (Table S1). Duplication events are related to plant evolutionary patterns. In this study, thirty segmentally duplicated genes were identified, and no tandem duplicated gene was found (Figure 4). All the synteny gene pairs were in the same cluster group. The substitution rates of Ka (non-synonymous) and Ks (synonymous) were calculated to estimate the selection pressure and determine the divergence time of the duplicated events. A total of 20 segmentally duplicated gene pairs were identified from

38 *OfXTHs* (Table 2). Interestingly, the Ka/Ks ratios of these 20 *OfXTHs* segmentally duplicated gene pairs were less than 1, suggesting that all segmentally duplicated gene pairs were purified selected instead of positively selected. The divergence time of *OfXTH* genes suggested that the duplicated events dated back to 59.93 million years ago (Mya) and lasted up to 0.31 Mya, and most of the duplicated events occurred between 5 and 35 Mya (Table 2).

**Figure 3.** Chromosomal distribution of *OfXTH* genes.




*OfXTH12-OfXTH22* 0.130 0.806 0.161 Purifying 26.87 *OfXTH13-OfXTH38* 0.038 0.194 0.194 Purifying 6.48 *OfXTH16-OfXTH19* 0.000 0.009 0.000 Purifying 0.31 *OfXTH18-OfXTH32* 0.143 0.958 0.150 Purifying 31.92 *OfXTH24-OfXTH31* 0.123 1.094 0.112 Purifying 36.48 *OfXTH25-OfXTH30* 0.078 0.345 0.226 Purifying 11.49 *OfXTH26-OfXTH37* 0.171 0.526 0.326 Purifying 17.54 *OfXTH27-OfXTH36* 0.038 0.222 0.169 Purifying 7.40

**Divergence Time (Mya)**

**Table 2.** *Cont.*

**Figure 4.** Synteny analysis of 38 *OfXTH* genes in *O. fragrans*. Orange rectangles represent chromosomes of *O. fragrans*, and blue lines represent segment duplicate gene pairs.

## *2.5. Analysis of Cis-Acting Regulatory Elements in the Promoters of OfXTH Genes*

For further understanding of the cis-acting regulatory elements in the promoter of *OfXTHs*, 2 kb upstream sequences of the transcription initiation site were subjected to Plant-CARE. A large number of cis-acting elements in response to hormones were found in *OfXTH* promoter sequences (Figure 5), including ABA (abscisic acid), MeJA (methyl jasmonate), GA (gibberellin), SA (salicylic acid), and auxin-responsive elements. Except for *OfXTH15* and *OfXTH32*, at least one ABA-responsive element was found in other *OfXTH* promoter sequences. Cis-acting regulatory elements involved in MeJA were presented in 29 *OfXTHs*. In addition, other cis-acting regulatory elements, including anaerobic induction, drought inducibility, low temperature, etc., were found in 33, 26, and 11 *OfXTH* promoters, respectively.

**Figure 5.** Prediction of cis-responsive elements in the *OfXTH* promoters. Different cis-responsive elements are represented by different colored boxes.

#### *2.6. OfXTH Genes Expression Analysis during Flower-Opening Stages*

To understand which *OfXTH* genes are involved in the regulation of the flowering processes, the quantitative real-time PCR was performed to detect *OfXTH* expression patterns during different flowering stages in *O. fragrans*. Flower bud phenotype at different stages are shown in Figure S1. According to the expression profiles, *OfXTHs* can be classified into three major groups (Figure 6). The first group, including four genes (*OfXTH8*, *12*, *26* and *37*), exhibited that these *OfXTH* genes were highly expressed in the S0 period and then decreased at other flowering stages (Figure 6A). Twenty-eight genes belong to the second group, including *OfXTH1*–*7*, *9*–*15*, *17*, *21*–*24*, *27*–*32*, *34*–*36* and *38*. The expression pattern analysis showed that these *OfXTH* genes significantly upregulated in the S1 and S2 period, or only expressed in the S2 period (Figure 6B). Finally, six *OfXTH* genes were categorized into the third group, including *OfXTH16*, *18*–*20*, *25* and *33*. This group of genes have a high expression level in flowering periods S4 and S5 (Figure 6C).
