Integrated RNA-Seq and DAP-Seq Analyses Identify a DntMYB1-Centered Regulatory Module Controlling Purple Flower...

Flower color is a key ornamental trait in Nobile-type Dendrobium, yet the molecular basis underlying purple flower formation remains poorly understood. In this study, a white-flowered paternal cultivar and its purple-flowered filial line were used to investigate the regulatory mechanism of purple floral pigmentation. Comparative phenotypic analysis showed that floral color divergence was established early during flower development, and anthocyanidin profiling of full-bloom petals revealed that purple flowers accumulated substantially higher levels of most anthocyanidins than white flowers, with delphinidin- and cyanidin-derived anthocyanidins together accounting for 80.26% and 94.56% of the total anthocyanidins in white- and purple-flowered materials, respectively. Transcriptome profiling identified a total of 21,235 differentially expressed genes (DEGs), with significant enrichment of phenylpropanoid- and flavonoid-related pathways, and MYB transcription factors prominently represented among the candidate regulators across the three comparison groups. Among them, DntMYB1 was identified as a C1 subgroup R2R3-MYB associated with floral pigmentation, and transient overexpression assays in Phalaenopsis hybrid V3 and a Nobile-type Dendrobium hybrid supported its positive role in visible purple pigmentation. By integrating RNA-seq and DAP-seq analyses, we identified 3205 candidate downstream targets of DntMYB1 and established a DntMYB1-centered regulatory module. Among these candidates, one Dnt4CL1 gene and one DntF3'H1 gene were validated as robust direct targets of DntMYB1 through yeast one-hybrid, EMSA, and dual-luciferase assays. These findings suggest that DntMYB1 is associated with purple flower formation by coordinately regulating both upstream precursor metabolism and downstream anthocyanin biosynthesis, providing new insight into the molecular regulation of flower color in Nobile-type Dendrobium and useful candidate genes for ornamental trait improvement.