*2.3. Multiple Sequence Alignment and Structure Analysis*

The WRKYGQK sequence is a considerably conservative motif of WRKY proteins and several variants of this conserved WRKY motif have been reported in plants [36], including WRKYGEK, WRKYGKK, WSKYEQK, and WRKYSEK. In the present study, this motif was observed in all longan WRKY proteins and three variants of this motif were also found. The majority of DlWRKY proteins contained the WRKYGQK motif, and WRKYGKK and WKKYRQK were observed in DlWRKY19 and DlWRKY47, respectively. The other remarkably conservative motif was a zinc finger structure which contained two types of zinc finger motifs: C–X4-5–C–X22–23–HXH and C–X7–C–X23–HXC. A total of 46 DlWRKY proteins contained C–X4-5–C–X22–23–HXH, and nine DlWRKY proteins contained C–X7–C–X23–HXC, which all belonged to Group III (Table S1).

According to the Gene Structure Display Server (GSDS) website, the number of introns was in the range of 1–5 in all the longan WRKY gene families, with most of *DlWRKY* genes containing 2–4 introns (*n* = 81.0%). The average number of introns was 2.82. In addition, the phylogenetic analysis of the *DlWRKY* gene family showed that the genes within the same group generally exhibited a similar exon/intron structure. For example, subgroup IIe contained two introns (Figure 2).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 5 of 20

**Figure 1.** The phylogenetic analysis of the longan WRKY proteins with orthologous members from grape and *Arabidopsis.* The maximum likelihood phylogenetic tree was constructed by MEGA 6.0. Different groups of DlWRKY proteins are indicated by a circle and the different colors. **Figure 1.** The phylogenetic analysis of the longan WRKY proteins with orthologous members from grape and *Arabidopsis.* The maximum likelihood phylogenetic tree was constructed by MEGA 6.0. Different groups of DlWRKY proteins are indicated by a circle and the different colors. **Figure 1.** The phylogenetic analysis of the longan WRKY proteins with orthologous members from grape and *Arabidopsis.* The maximum likelihood phylogenetic tree was constructed by MEGA 6.0. Different groups of DlWRKY proteins are indicated by a circle and the different colors.

The phylogenetic tree was constructed by MEGA 6.0. The red color indicates the untranslated 5′ and 3′-regions; the blue color indicates exons; and the gray color indicates introns. **Figure 2.** The unrooted phylogenetic tree (**left**) and gene structure (**right**) of 55 DlWRKY proteins. The phylogenetic tree was constructed by MEGA 6.0. The red color indicates the untranslated 5′ and 3′-regions; the blue color indicates exons; and the gray color indicates introns. **Figure 2.** The unrooted phylogenetic tree (**left**) and gene structure (**right**) of 55 DlWRKY proteins. The phylogenetic tree was constructed by MEGA 6.0. The red color indicates the untranslated 50 - and 3 0 -regions; the blue color indicates exons; and the gray color indicates introns.

To further understand the similarity and diversity of motif composition among different DlWRKY proteins, a phylogenetic tree based on the full-length DlWRKY proteins was constructed (Figure 3). The motifs in the DlWRKY protein sequences were also predicted using MEME (http://meme.sdsc. edu/meme/cgi-bin/meme.cgi) (Figure 3 and Table S2). A total of 15 motifs were identified to illustrate the WRKY protein structure in longan. The results showed that the number of motifs in DlWRKYs ranged from 2 to 6, and the length of motifs ranged from 21 to 50 amino acids. Among the 15 identified motifs, motifs 1 and 2, characterized as WRKY domains, were broadly distributed across the DlWRKYs. To further understand the similarity and diversity of motif composition among different DlWRKY proteins, a phylogenetic tree based on the full-length DlWRKY proteins was constructed (Figure 3). The motifs in the DlWRKY protein sequences were also predicted using MEME (http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) (Figure 3 and Table S2). A total of 15 motifs were identified to illustrate the WRKY protein structure in longan. The results showed that the number of motifs in DlWRKYs ranged from 2 to 6, and the length of motifs ranged from 21 to 50 amino acids. Among the 15 identified motifs, motifs 1 and 2, characterized as WRKY domains, were broadly distributed across the DlWRKYs.

**Figure 3.** The unrooted phylogenetic tree (**left**) and conserved motifs (**right**) of 55 DlWRKY proteins. The phylogenetic tree was constructed using the same method used in Figure 2. Different colors represent various groups. MEME was used to predict motifs, and these motifs are represented by boxes. **Figure 3.** The unrooted phylogenetic tree (**left**) and conserved motifs (**right**) of 55 DlWRKY proteins. The phylogenetic tree was constructed using the same method used in Figure 2. Different colors represent various groups. MEME was used to predict motifs, and these motifs are represented by boxes.
