Characterization and transcriptomic analysis provide novel insights into the sexual differentiation of the dioecious plant Bama Huoma (Cannabis sativa).

Background: Bama Huoma is a high-value cash crop because its seeds are known as the "holy seeds of longevity". However, its seed yield is restricted by dioecism, and information about flower sex differentiation in this plant is lacking.

Results: In this study, anatomical analysis and dynamic transcriptome profiling were performed to explore the mechanism of sex differentiation in Bama Huoma. The results revealed that female and male flowers undergo independent developmental processes and that sex differentiation occurs at an early stage before flower development. Illumina sequencing revealed that MADS-box genes play crucial roles in pistil and stamen development. qRT‒PCR and RNA in situ hybridization were subsequently performed to determine the spatiotemporal expression patterns of genes related to sex determination. The results revealed that eight MADS-box genes and two MYB genes were expressed in a sex-biased and tissue-specific manner in male or female flowers. Among these genes, MYB35, PMADS2, TM6 and AGL104 were specifically expressed in male flowers and found to regulate callose metabolism, pollen sac formation, tapetum development, and microspore maturation. In contrast, AGL11, FBP24, AGL16 and two CAL were restricted to female flowers, where they regulate pistil formation, ovary development, and stigma differentiation. These genes play distinct and essential roles in the sex differentiation of Bama Huoma, as well as in the development of floral organs.

Conclusion: Our findings provide a comprehensive gene expression profile of flower sex differentiation in Bama Huoma as well as new clues and information for further study in this field.