*2.4. Tissue-Specific Expression Patterns of DlWRKY 2.4. Tissue-Specific Expression Patterns of DlWRKY*

two or three tissues.

To generate expression profiles of *DlWRKY* genes under normal conditions, the expression levels of the 55 *DlWRKY* genes in the root, stem, leaf, seed, young fruit, pulp, pericarp, flower, and flower bud were investigated by the RNA-seq analysis. The log10 (FPKM + 0.01) values of the transcripts were clustered hierarchically and displayed in a heat map (Figure 4 and Table S3). The results showed that 96.36% (53 of 55) of *DlWRKYs* were expressed in young fruits and 94.55% were expressed in the pericarp, stems, and flower bud. A total of 90.91%, 89.09%, and 81.82% of *DlWRKYs* were expressed in the flower, leaf, root, and seed, respectively. Only a few *DlWRKY* genes were detected in pulps (67.27%). Approximately 60% (33 of 55) of the *DlWRKY* genes were expressed in each tested tissue, in which 25 *DlWRKY* genes (*DlWRKY*1, 2, 3, 5, 6, 8, 9, 13, 14, 23, 24, 28, 30, 32, 35, 37, 38, 39, 44, 49, 50, 52, 53, and 54) were highly expressed in at least six longan tissues. In contrast, 12 *DlWRKY* genes *(DlWRKY*10, 12, 18, 22, 26, 36, 40, 41, 42, 45, 47, and 48) were expressed at low levels in all tested tissues. Furthermore, *DlWRKY*22 only displayed a significantly low expression in the flower bud. *DlWRKY*10, 22, 41, 47, and 48 were preferential accumulation in To generate expression profiles of *DlWRKY* genes under normal conditions, the expression levels of the 55 *DlWRKY* genes in the root, stem, leaf, seed, young fruit, pulp, pericarp, flower, and flower bud were investigated by the RNA-seq analysis. The log<sup>10</sup> (FPKM + 0.01) values of the transcripts were clustered hierarchically and displayed in a heat map (Figure 4 and Table S3). The results showed that 96.36% (53 of 55) of *DlWRKYs* were expressed in young fruits and 94.55% were expressed in the pericarp, stems, and flower bud. A total of 90.91%, 89.09%, and 81.82% of *DlWRKYs* were expressed in the flower, leaf, root, and seed, respectively. Only a few *DlWRKY* genes were detected in pulps (67.27%). Approximately 60% (33 of 55) of the *DlWRKY* genes were expressed in each tested tissue, in which 25 *DlWRKY* genes (*DlWRKY*1, 2, 3, 5, 6, 8, 9, 13, 14, 23, 24, 28, 30, 32, 35, 37, 38, 39, 44, 49, 50, 52, 53, and 54) were highly expressed in at least six longan tissues. In contrast, 12 *DlWRKY* genes *(DlWRKY*10, 12, 18, 22, 26, 36, 40, 41, 42, 45, 47, and 48) were expressed at low levels in all tested tissues. Furthermore, *DlWRKY*22 only displayed a significantly low expression in the flower bud. *DlWRKY*10, 22, 41, 47, and 48 were preferential accumulation in two or three tissues.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 7 of 20

**Figure 4.** The heat map of the *DlWRKY* gene expression profiles in different tissues. The color scale represents the log10 expression values; the red and green colors indicate the higher or lower transcript abundances compared to the relevant control, respectively. **Figure 4.** The heat map of the *DlWRKY* gene expression profiles in different tissues. The color scale represents the log<sup>10</sup> expression values; the red and green colors indicate the higher or lower transcript abundances compared to the relevant control, respectively.

#### *2.5. Comparative Expression Profiles of Two Longan Species during the Flowering Process*  Although the involvement of many WRKY genes has been examined in the control of *2.5. Comparative Expression Profiles of Two Longan Species during the Flowering Process*

flowering time [15], the expression of *DlWRKY* genes during flower induction has not been studied extensively. In the present study, we also analyzed the expression patterns of 55 *DlWRKY* genes in two longan species during the three flowering stages by RNA-seq analysis (Table S4). Heat maps were constructed based on the log10 (FPKM + 0.01) values for the 55 *DlWRKY* genes (Figure 5a). Based on the criteria for *p*-values <0.05 and fold changes ≥2, the *DlWRKY* genes that were differentially expressed during the three flowering stages of the two longan species were identified. Interestingly, the results showed that all 55 *DlWRKY* genes were constructively expressed in the three test flowering stages of the "SX" longan, while 18 *DlWRKY* genes showed a specific expression in the "SJ" longan. Among the 18 *DlWRKY* genes, 12 (*DlWRKY*5, 7, 8, 9, 15, 21, 23, 24, 25, 39, 52, and 54) showed a continuously down-regulated expression through the three flowering stages, and four genes (*DlWRKY*16, 17, 41, and 42) showed an up-regulated expression. Moreover, two genes (*DlWRKY*10 and 48) showed a transient up-regulation at the second stage and a down-regulation at the third stage. To validate the expression levels obtained from the RNA-seq data, twelve *DlWRKY* genes Although the involvement of many WRKY genes has been examined in the control of flowering time [15], the expression of *DlWRKY* genes during flower induction has not been studied extensively. In the present study, we also analyzed the expression patterns of 55 *DlWRKY* genes in two longan species during the three flowering stages by RNA-seq analysis (Table S4). Heat maps were constructed based on the log<sup>10</sup> (FPKM + 0.01) values for the 55 *DlWRKY* genes (Figure 5a). Based on the criteria for *p*-values <0.05 and fold changes ≥2, the *DlWRKY* genes that were differentially expressed during the three flowering stages of the two longan species were identified. Interestingly, the results showed that all 55 *DlWRKY* genes were constructively expressed in the three test flowering stages of the "SX" longan, while 18 *DlWRKY* genes showed a specific expression in the "SJ" longan. Among the 18 *DlWRKY* genes, 12 (*DlWRKY*5, 7, 8, 9, 15, 21, 23, 24, 25, 39, 52, and 54) showed a continuously down-regulated expression through the three flowering stages, and four genes (*DlWRKY*16, 17, 41, and 42) showed an up-regulated expression. Moreover, two genes (*DlWRKY*10 and 48) showed a transient up-regulation at the second stage and a down-regulation at the third stage.

(*DlWRKY*1, 5, 9, 15, 16, 17,18,24,39,42, 48, and 50) were selected from the six different longan WRKY groups for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. Consistent with the result of the RNA-seq analysis, the transcript levels of all twelve *DlWRKY* genes did not exhibit any significant differences in the "SX" longan between the three flowering stages (Figure 5b). In addition, the relative expression level of *DlWRKY*1, *DlWRKY*18, and *DlWRKY*50 did not exhibit any significant differences in '""SJ" during the three flowering stages. The expression levels of *DlWRKY*16, 17, 42, and 48were up-regulated in the second and third stage. To validate the expression levels obtained from the RNA-seq data, twelve *DlWRKY* genes (*DlWRKY*1, 5, 9, 15, 16, 17,18,24,39,42, 48, and 50) were selected from the six different longan WRKY groups for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. Consistent with the result of the RNA-seq analysis, the transcript levels of all twelve *DlWRKY* genes did not exhibit any significant differences in the "SX" longan between the three flowering stages (Figure 5b). In addition, the relative expression level of *DlWRKY*1, *DlWRKY*18, and *DlWRKY*50 did not exhibit any significant differences in '""SJ" during the three flowering stages. The expression levels of *DlWRKY*16, 17, 42, and 48were up-regulated in the second and third stage. The transcript level of *DlWRKY*5, 9, 15, 24, and *DlWRKY*39 was down-regulated in the second and third stages (Figure 5b). In general, the expression levels obtained by qRT-PCR for these genes are similar to the results obtained from the RNA-seq data. The transcript level of *DlWRKY*5, 9, 15, 24, and *DlWRKY*39 was down-regulated in the second and third stages (Figure 5b). In general, the expression levels obtained by qRT-PCR for these genes are similar to the results obtained from the RNA-seq data.

**Figure 5.** *Cont.*

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**Figure 5.** The expression profiles of *DlWRKY* in two longan species during the floral induction process. (**a**) A heat map showing the comparative expression level of the *WRKY* genes in the three flowering stages of"SJ" and "SX". The color scale represents the log10 expression values. Genes with comparatively low expression values are shown using shades of green, and high expression values are represented using shades of red. The three flowering stages of SJ are indicated by SJT1, SJT2, and SJT3. The three flowering stages of SX are indicated by SXT1, SXT2, and SXT3. (**b**) Relative expression levels of the twelve *DlWRKYs* during the three flowering stages of the two longan species by qRT-PCR. For each gene, the relative expression level in T1 (dormant apical bud) was set as one, and the longan *actin* gene was used as the internal expression control. The data represent the mean ± SD of the three replicates. Values with the same letter were not significantly different when assessed using Duncan's multiple range test (*p* < 0.05, *n* = 3). **Figure 5.** The expression profiles of *DlWRKY* in two longan species during the floral induction process. (**a**) A heat map showing the comparative expression level of the *WRKY* genes in the three flowering stages of"SJ" and "SX". The color scale represents the log<sup>10</sup> expression values. Genes with comparatively low expression values are shown using shades of green, and high expression values are represented using shades of red. The three flowering stages of SJ are indicated by SJT1, SJT2, and SJT3. The three flowering stages of SX are indicated by SXT1, SXT2, and SXT3. (**b**) Relative expression levels of the twelve *DlWRKYs* during the three flowering stages of the two longan species by qRT-PCR. For each gene, the relative expression level in T1 (dormant apical bud) was set as one, and the longan *actin* gene was used as the internal expression control. The data represent the mean ± SD of the three replicates. Values with the same letter were not significantly different when assessed using Duncan's multiple range test (*p* < 0.05, *n* = 3).
