Genome-Wide Identification of the WRKY Gene Family in Mulberry and Screening of Consistently Drought-Responsive Candidates

Abstract

Background: WRKY transcription factors are widely involved in plant responses to drought. Mulberry (Morus alba) is an economically important tree in Southwest China, yet its WRKY gene family has not been systematically characterized at the genome level. Methods: In this study, we identified the WRKY gene family in mulberry by integrating bioinformatic analysis with transcriptome data and experimental verification. Results: A total of 56 MnWRKY genes were identified and classified into three major groups (I–III), with group II further divided into five subgroups (IIa–IIe). Promoter analysis detected 577 cis-acting elements belonging to ten representative categories, among which ABA-responsive elements were the most abundant. Chromosomal localization showed that 73.2% of the genes were concentrated on the first three chromosomes, and collinearity analysis identified three tandem and 21 segmental duplication pairs. By integrating public RNA-seq data with our transcriptome data from the drought-tolerant cultivar Yunsang-2, we found eight MnWRKY genes (MnWRKY4, MnWRKY8, MnWRKY9, MnWRKY11, MnWRKY13, MnWRKY18, MnWRKY20, and MnWRKY21) that were consistently upregulated under drought in both datasets. qRT-PCR further confirmed that MnWRKY9 and MnWRKY18 were significantly induced by drought, with MnWRKY9 showing the strongest response. Transient expression in tobacco leaves showed that MnWRKY9 localizes to the nucleus. Conclusions: MnWRKY9 is identified as a candidate regulator of drought response in mulberry. These results provide a useful resource for further functional studies of WRKY genes and for molecular breeding of drought-tolerant mulberry varieties.