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The WRKY transcription factor family plays important regulatory roles in multiple biological processes in higher plants. They have been identified and functionally characterized in a number of plant species, but very little is known in Neolamarckia cadamba, a 'miracle tree' for its fast growth and potential medicinal resource in Southeast Asia. In this study, a total of 85 WRKY genes were identified in the genome of N. cadamba. They were divided into three groups according to their phylogenetic features, with the support of the characteristics of gene structures and conserved motifs of protein. The NcWRKY genes were unevenly distributed on 22 chromosomes, and there were two pairs of segmentally duplicated events. In addition, a number of putative cis-elements were identified in the promoter regions, of which hormone- and stress-related elements were shared in many NcWRKYs. The transcript levels of NcWRKY were analyzed using the RNA-seq data, revealing distinct expression patterns in various tissues and at different stages of vascular development. Furthermore, 16 and 12 NcWRKY genes were confirmed to respond to various hormone treatments and two different abiotic stress treatments, respectively. Moreover, the content of cadambine, the active metabolite used for the various pharmacological activities found in N. cadamba, significantly increased after Methyl jasmonate treatment. In addition, expression of NcWRKY64/74 was obviously upregulated, suggesting that they may have a potential function of regulating the biosynthesis of cadambine in response to MeJA. Taken together, this study provides clues into the regulatory roles of the WRKY gene family in N. cadamba. [ABSTRACT FROM AUTHOR]