Abstract
Cyperus rotundus, a globally distributed and highly competitive weed, has evolved herbicide resistance, posing a significant challenge to sustainable agriculture; however, the lack of genomic resources has limited comprehensive investigations into its resistance mechanisms. Here, we report a chromosome-level genome assembly of triploid C. rotundus (875.13 Mb across 165 chromosomes), which exhibits high synteny among haplotypes. Comparative analysis of six populations revealed that only a population from Changde, Hunan, China (designated R-HN) displayed dual resistance to glyphosate and glufosinate. In this population, high expression of the target-site resistance gene glutamine synthetase 2 (GS2) contributes to glufosinate resistance, whereas glyphosate resistance is predominantly mediated by non-target-site resistance (NTSR) mechanisms. By integrating transcriptomic profiling with yeast-based functional validation, we identified two NTSR genes, CrABCG15 and CrCASPL2C2, that confer glyphosate resistance. Collectively, this study provides a high-quality genomic resource for C. rotundus and advances our understanding of its genomic evolution and herbicide resistance mechanisms.