Despite extensive research on orchid reproductive strategies, the genetic studies of sex differentiation in the orchid family are still lacking. In this study, we compared three sexual phenotypes of Cymbidium tortisepalum bisexual flowers as well as female and male unisexual mutants. Through comparative transcriptomes, we analyzed the sex-biased differentially expressed genes (DEGs) and gene co-expression networks of sex organs (gynostemium and ovary) among them, identified the candidate genes of sex differentiation, and validated their expression by qRT-PCR. The C. tortisepalum unisexual mutants with degenerated phenotypes were compared to the bisexual plants with respect to both the flower organs and plant morphologies. Totally, 12,145, 10,789, and 14,447 genes were uniquely expressed in the female, male, and hermaphrodite sex organs, respectively. A total of 4291 sex-biased DEGs were detected among them, with 871, 2867, and 1937 DEGs in the comparisons of bisexual vs. female, bisexual vs. male, and male vs. female flowers, respectively. Two co-expressed network modules, with 81 and 419 genes were tightly correlated with female sexual traits, while two others with 265 and 135 genes were highly correlated with male sexual traits. Two female-biased hub genes (CtSDR3b and CtSDR3b-like) nested in the female modules, the homologs of maize sex determinant tasselseed2, may control the feminization of C. tortisepalum. At the same time, two male-biased hub genes (CtYAB2 and CtYAB5) nested in the male modules, the homologs of grape sex determinant VviYABBY3, may control the androphany of C. tortisepalum. This study discovered the molecular regulation networks and proposed a model for orchid sex differentiation, therefore providing for the first time the genetic basis for the sex separation in the orchid family.

Spinach is a beloved vegetable crop and widely cultivated worldwide. It is dioecious with male and female plants, although monoecious mutations exist. Female spinach exhibits two distinct sepal morphologies—thorn-shaped and round-shaped determine seed shape and strongly influence seed handling, mechanized sowing, and cultivar classification. To dissect the genetic basis of this trait, we developed an F₂ population from contrasting parental lines and constructed a high-density linkage map with ~1615 bin markers spanning ~994.04 cm. A major 4.31 Mb genomic interval on chromosome 4, flanked by tightly linked markers, was consistently associated with sepal morphology. Transcriptome profiling across early and mature sepal developmental stages revealed significant enrichment of cell cycle-related pathways, including DNA replication, repair, mitosis, and cytokinesis. By integrating differential expression analysis with weighted gene co-expression network analysis, we identified 25 DEGs within the mapped interval, 11 of which showed strong co-expression with hub genes in trait-associated modules. Structural variation analysis further uncovered promoter and coding sequence polymorphisms in a subset of candidate genes. This study highlights 11 promising candidate genes potentially regulating sepal-derived seed morphology in spinach, rather than confirming definitive causal genes, providing valuable targets for functional validation and new insights into the genetic regulation of sepal development.

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 resistance, and ornamental and agronomic value in orchids.