目的 针对大麻Cannabis sativa早期性别鉴定困难及现有分子标记稳定性不足的问题，基于大麻性染色体结构特征，开发稳定、准确的大麻早期性别鉴定分子标记，为生产中的幼苗期性别鉴定提供可靠技术手段。方法 综合利用多份染色体级别大麻单倍型基因组数据，通过生物信息学方法系统比对筛选Y染色体特异区段（MSY）中的雄性特异片段；从大片段MSY区域设计特异性分子标记，以36份已知性别大麻样品（包括15份雄株和21份雌株）为材料，通过PCR扩增及琼脂糖凝胶电泳，对分子标记进行特异性与稳定性验证，并与已报道标记（MADC5、MADC6）进行对比分析。结果 共筛选获得15 648个Y染色体的特异区段，从中开发出12个SCAR标记。经验证，其中5个标记（MSY99M-3、MSY99M-4、MSY99M-5、MSY100M-1、MSY100M-7）在雄株中均能稳定扩增出清晰特异性条带，而在所有雌株中均无扩增，鉴定准确率达100%，优于部分已报道标记。结论 基于Y染色体特异区段开发的SCAR标记具有高度特异性和稳定性，可用于大麻早期性别的快速、精准鉴定，对提高药用大麻相关产品的生产效率具有重要的应用价值。

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.

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国家自然科学基金青年基金项目（82204579）；中央级公益性科研院所基本科研业务费专项（ZZ15-YQ-039，ZXKT22051）