Sex chromosome-aware GWAS reveals XY-linked agronomic architecture and breeding targets in cannabis

Cannabis sativa is a dioecious crop whose male plants carry a Y chromosome entirely excluded from every genome-wide association study (GWAS) published to date, despite its direct relevance to agronomic trait variation. Here, we present the first sex chromosome-aware GWAS of cannabis, integrating both ChrX and ChrY across 145 landrace accessions and 27 agronomic traits. By combining SNP-based GWAS with sex-stratified k-mer association analysis, we identified 16 genome-wide significant markers: eight on ChrX controlling traits including stem biomass (CsWSL1; phenotypic variance explained (PVE) 56.4%) and relative growth rate (CsX8; PVE 73.6%), and eight on ChrY controlling male morphology (CsMSML1; PVE 17.18-76.1%) and male-specific flowering (CsMSFL1; PVE 84.08-87.1%). K-mer GWAS yielded 1,838 annotated signal-trait associations representing 1,082 unique genomic windows across the pseudoautosomal, hemizygous X, and male-specific Y regions. The key loci identified here provide a genomic toolkit for marker-assisted paternal selection, flowering-time synchronization, and biomass improvement across dioecious cannabis breeding systems. HIGHLIGHTFirst sex chromosome-aware GWAS of cannabis integrating ChrX and ChrY identifies 16 agronomic loci including CsMSFL1, a near-deterministic regulator of male flowering with 87% phenotypic variance explained.