| Summary: | Class III homeodomain-leucine zipper (HD-ZIP III) genes encode plant-specific transcription factors that play pivotal roles in plant growth and development. There is no systematic report on HD-ZIP III members in <i>Brassica</i> plants and their responses to stress are largely unknown. In this study, a total of 10, 9 and 16 HD-ZIP III genes were identified from <i>B. rapa</i>, <i>B. oleracea</i> and <i>B. napus</i>, respectively. The phylogenetic analysis showed that HD-ZIP III proteins were grouped into three clades: PHB/PHV, REV and CNA/HB8. Genes in the same group tended to have similar exon–intron structures. Various phytohormone-responsive elements and stress-responsive elements were detected in the promoter regions of HD-ZIP III genes. Gene expression levels in different tissues, as well as under different stress conditions, were investigated using public transcription profiling data. The HD-ZIP III genes were constitutively expressed among all the tested tissues and were highly accumulated in root and stem. In <i>B. rapa</i>, only one <i>BrREV</i> gene especially responded to heat stress, <i>BrPHB</i> and <i>BrREV</i> members were downregulated upon cold stress and most HD-ZIP III genes exhibited divergent responses to drought stress. In addition, we investigated the genetic variation at known miR165/166 complementary sites of the identified HD-ZIP III genes and found one single nucleotide polymorphism (SNP) in <i>PHB</i> members and two SNPs in <i>REV</i> members, which were further confirmed using Sanger sequencing. Taken together, these results provide information for the genome-wide characterization of HD-ZIP III genes and their stress response diversity in <i>Brassica</i> species.
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