Pan-Genomic Evolution of R2R3-MYB and bHLH Transcription Factors in Dendrobium

R2R3-MYB and bHLH transcription factors (TFs) are key regulators of floral secondary metabolism and epidermal development in flowering plants. Orchids exhibit remarkable floral diversity, which is critical for pollination success and ornamental value, yet the evolutionary and functional diversification of these TF families within the genus remains largely unexplored. Here, we conducted a comprehensive pan-genome dissection of R2R3-MYB and bHLH TF families across 18 Dendrobium species, integrating orthologs assignment, phylogenetics, duplication profiling, cis-regulatory annotation, and tissue-specific expression analysis. We identified 3074 R2R3-MYB and 2282 bHLH genes, classified into 69 and 61 orthologous gene groups (OGGs), respectively. Core OGGs accounted for two-thirds of both families, indicating strong evolutionary conservation, whereas variable OGGs reflected lineage-specific diversification. Phylogenetic analyses resolved R2R3-MYBs into 24 canonical subfamilies and revealed conserved heterogeneous expansion patterns in bHLH subfamilies. Promoter architectures of R2R3-MYB genes were enriched in hormone-, stress-, and light-responsive elements, whereas bHLH promoters were dominated by development-related motifs. Tissue-specific expression profiling in Dendrobium ‘Chao Praya Smile’ showed that floral bud-enriched genes were associated with flavonoid/anthocyanin biosynthesis, whereas root-enriched genes were linked to stress and hormone responses. Integration of pan-genomics and transcriptomics highlighted evolutionary trajectory and functional divergence between core and variable gene sets within Dendrobium. Our study establishes a comprehensive, genus-wide framework for understanding the evolutionary and functional characteristics of MYB–bHLH regulatory networks in Dendrobium. These findings provide valuable genetic resources and key candidate targets for functional characterization and molecular breeding, with important implications for genetic improvement of reproductive traits, floral quality, stress ...