Objective To address the difficulty of early sex identification in Cannabis sativa and the instability of existing molecular markers, this study developed stable and accurate sex identification molecular markers based on the structural characteristics of sex chromosomes of C. sativa. This approach aims to provide a reliable tool for early sex determination at the seedling stage. Methods By comprehensively utilizing multiple chromosome-level haplotype genomic data of C. sativa, the male-specific segments in the male-specific of Y (MSY) region were systematically compared and screened through bioinformatics methods. Specific molecular markers were then designed from large MSY fragments and tested for specificity and stability using 36 C. sativa samples with known sex (15 males and 21 females) through PCR amplification and agarose gel electrophoresis, and the newly developed markers were compared with previously reported ones (MADC5 and MADC6) for validation. Results A total of 15 648 Y chromosome-specific sequences were identified, from which 12 SCAR markers were developed. Among them, five markers (MSY99M-3, MSY99M-4, MSY99M-5, MSY100M-1, and MSY100M-7) consistently produced distinct male-specific bands in all male plants, without amplification in female samples, achieving 100% identification accuracy, which was superior to that of previously reported markers. Conclusion The SCAR markers developed from Y chromosome-specific regions demonstrated high specificity, stability, and relia, enabling rapid and precise early sex identification in C. sativa. These markers offer significant potential to enhance the production efficiency of medicinal cannabis and related products.

Cannabis sativa L.  /  sex identification  /  molecular marker  /  Y chromosome  /  SCAR marker