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Drought is a major limiting factor affecting grain production. Drought-tolerant crop varieties are required to ensure future grain production. Here, 5597 DEGs were identified using transcriptome data before and after drought stress in foxtail millet (Setaria italica) hybrid Zhangza 19 and its parents. A total of 607 drought-tolerant genes were screened through WGCNA, and 286 heterotic genes were screened according to the expression level. Among them, 18 genes overlapped. One gene, Seita.9G321800, encoded MYBS3 transcription factor and showed upregulated expression after drought stress. It is highly homologous with MYBS3 in maize, rice, and sorghum and was named SiMYBS3. Subcellular localization analysis showed that the SiMYBS3 protein was located in the nucleus and cytoplasm, and transactivation assay showed SiMYBS3 had transcriptional activation activity in yeast cells. Overexpression of SiMYBS3 in Arabidopsis thaliana conferred drought tolerance, insensitivity to ABA, and earlier flowering. Our results demonstrate that SiMYBS3 is a drought-related heterotic gene and it can be used for enhancing drought resistance in agricultural crop breeding.

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