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Anthocyanins are responsible for the intensity of color in plants; however, the systematic mechanisms underlying the color differences in the fruit of Ailanthus altissima remain unknown. Therefore, this study aims to analyze the transcriptomes of the white and red fruit of A. altissima by screening and validating the key genes involved in flavonoid and anthocyanin biosynthesis. Samples of A. altissima fruit were collected 30, 45, and 60 days after flowering, and their pigment and sugar content were determined. The anthocyanin content was significantly higher in red than in white fruits. Transcriptome analysis was also performed on the fruit samples, 73,807 unigenes were assembled and annotated to seven databases. Twenty‐one co‐expressed modules were identified via weighted gene co‐expression network analysis, of which two were associated with flavonoids and anthocyanins. Furthermore, in three growth stages, 126, 30, and 124 differentially expressed genes were screened between white and red fruit. Genes involved in flavonoid and anthocyanin metabolism were identified. AaDFR (A. altissima bifunctional dihydroflavonol 4‐reductase/flavanone 4‐reductase) and AaANS (A. altissima anthocyanidin synthase) were associated with flavonoid and anthocyanin metabolism. Members of the AaDFR and AaANS families were also identified, and their basic physicochemical characteristics, conserved domains, motif compositions, phylogenetics, and expression levels were analyzed. The overexpression of AaDFR and AaANS in transgenic Arabidopsis significantly increased the content of seed and foliar flavonoids and anthocyanins. The study elucidated the different mechanisms underlying fruit color development and provided insight into A. altissima plants breeding with commercially desirable properties.

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