*4.5. Stress and Hormonal Treatments and Expression Profiling Using qRT-PCR*

Twenty-seven one-year-old uniform grafted seedlings of "SJ", obtained from the South Subtropical Crops Research Institute of the Chinese Academy of Tropical Agricultural Science in Zhanjiang (110◦160E, 21◦100N) were used for stress and hormonal treatments. For hormone treatments, three seedlings were treated with methyl jasmonate (MeJA) or SA solution (100 µM) for 4 h at 28 ◦C, respectively. Meanwhile, three seedlings sprayed with water were used as a control. For heat and cold stresses, three samples were grown at 42 or 0 ◦C for 4 h, respectively, and three samples grown at 28 ◦C were used as a control. All the treatments were performed in a greenhouse. Six leaves were collected

from each seedling and all samples were immediately frozen in liquid nitrogen and stored at −80 ◦C for expression analysis.

According to the manufacturer's instructions, the total RNA was obtained by using the SuperFast RNA extraction kit (Hua Yue Yang Bio Co.). The first-strand cDNA was synthesized by reverse transcription of the total RNA (500 ng) using PrimeScriptRTase (TaKaRa Biotechnology, Dalian, China). Gene-specific primers were designed according to the *DlWRKY* gene sequences using Primer Premier 5.0 and checked using Blastn in NCBI (Table S8). In addition, the longan *Actin*1 gene (Dlo\_028674) was used as an internal control for normalization. qRT-PCR was conducted using the LightCycler® 480 Real-Time PCR System (Roche, Germany) and SYBR Green II PCR Master Mix (Takara, Dalian, China). The amplification program was as follows: 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 15 s, and 60 ◦C for 1 min. Each reaction was performed in three replicates. The relative expression levels of the candidate genes were calculated by the 2–∆∆*C*<sup>t</sup> method. The analysis included cDNA from the three biological samples for each tissue, and all the reactions were run in triplicates. In the comparative expression analysis of the *DlWRKY* genes, genes that were up- or down-regulated by at least two-fold were considered differentially expressed.
