*4.2. Sequence Alignment, Phylogenetic Analysis, and Cis-Elements in the Promoters*

The 72 *Arabidopsis* and 59 grape WRKY proteins described previously [29,38] were obtained from TAIR (http://www.arabidopsis.org/) and NCBI (http://www.ncbi.nlm.nih.gov/), respectively. By using Clustal X version 1.83, the WRKY protein sequences of *Arabidopsis* and longan were aligned for phylogenetic analysis. Based on this alignment, a bootstrapped ML (Maximum Likelihood) tree was constructed using MEGA (version 6.0) with the bootstrap test replicated 1000 times [61]. To assess

the phylogenetic relationships among the members of the longan *WRKY* gene family, a phylogenetic tree was prepared according to the alignment of only the longan proteins. All DlWRKY transcription factors were classified into subgroups based on their structural features and evolutionary relationships. The 1500-bp sequences upstream of the start codon of the candidate *DlWRKY* genes were extracted from the longan genome sequences. The PlantCARE software (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) was used for searching the cis-acting elements [62].
