The MYB transcription factor (TF) superfamily, one of the largest gene superfamilies, regulates a variety of physiological processes in plants. Although many MYB superfamily genes have been identified in plants, the MYB TFs in Chinese jujube (Ziziphus jujuba Mill.) have not been fully identified and characterized. Additionally, the functions of these genes remain unclear. In total, we identified 171 MYB superfamily genes in jujube and divided them into five subfamilies containing 99 genes of the R2R3-MYB subfamily, 58 genes of the MYB-related subfamily, four genes of the R1R2R3-MYB subfamily, one gene of the 4R-MYB subfamily, and nine genes of the atypical MYB subfamily. The 99 R2R3-MYB genes of jujube were divided into 35 groups, C1-C35, and the 58 MYB-related genes were divided into the following groups: the R-R-type, CCA1-like, I-box-binding-like, TBP-like, CPC-like, and Chinese jujube-specific groups. ZjMYB genes in jujube were well supported by additional highly conserved motifs and exon/intron structures. Most R1 repeats of MYB-related proteins comprised the R2 repeat and had highly conserved EED and EEE residue groups in jujube. Three tandem duplicated gene pairs were found on 12 chromosomes in jujube. According to an expression analysis of 126 ZjMYB genes, MYB-related genes played important roles in jujube development and fruit-related biological processes. The total flavonoid content of jujube fruit decreased as ripening progressed. A total of 93 expressed genes were identified in the RNA-sequencing data from jujube fruit, and 56 ZjMYB members presented significant correlations with total flavonoid contents by correlation analysis. Five pairs of paralogous MYB genes within jujube were composed of nine jujube MYB genes. A total of 14 ZjMYB genes had the same homology to the MYB genes of Arabidopsis and peach, indicating that these 14 MYB genes and their orthologs probably existed before the ancestral divergence of the MYB superfamily. We used a synteny analysis of MYB genes between jujube and Arabidopsis to predict that the functions of the ZjMYBs involve flavonoid/phenylpropanoid metabolism, the light signaling pathway, auxin signal transduction, and responses to various abiotic stresses (cold, drought, and salt stresses). Additionally, we speculate that ZjMYB108 is an important TF involved in the flavonoid metabolic pathway. This comprehensive analysis of MYB superfamily genes in jujube lay a solid foundation for future comprehensive analyses of ZjMYB gene functions.

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