Cyperus difformis is a globally problematic weed in rice fields, posing a significant threat to rice yield. While chemical herbicides are commonly used for its control, the species often escapes management due to its rapid evolution of resistance to widely used herbicides. To better understand the mechanisms underlying herbicide resistance, insights into the genetics and genomics of C. difformis are essential. In this study, we present a telomere-to-telomere genome assembly of C. difformis, generated by combining PacBio HiFi, Oxford Nanopore Technologies (ONT), MGI short reads and high-throughput chromatin conformation capture (Hi-C) technologies. The assembled genome spans 226 Mb with a scaffold N50 of 13.08 Mb. Utilizing Hi-C interaction data, 97.24% of the contigs were anchored to 18 chromosomes, with 35 telomeres successfully defined. Further analysis identified 75.73 Mb of repetitive sequences and 21,069 protein-coding genes, of which 91.8% (19,347 genes) were functionally annotated. This high-quality genome provides a valuable resource for studies in population genetics, phylogeny, comparative genomics, adaptive evolution, and functional genomics of C. difformis.

The transition from puddled-transplanted rice (Oryza sativa L.) (PTR) to direct-seeded rice (DSR) is gaining popularity in central China. In contrast, the PTR system is the most common practice in southwest China. Weeds are a major problem in the paddy fields of the DSR systems, and herbicides are widely used for weed control. However, the increased frequency and rate of herbicide use leads to the rapid evolution of resistance. Smallflower umbrella sedge (Cyperus difformis L.) is a troublesome weed species in rice fields of China and is usually controlled by the acetolactate synthase (ALS)-inhibiting herbicide bensulfuron-methyl. Here, we collected 32 C. difformis populations from DSR systems (Hunan Province) and PTR systems (Guangxi Province) and investigated their resistance to bensulfuron-methyl. Results revealed 80% (8 out of 10) populations from Hunan Province and 14% (3 out of 22) populations from Guangxi Province had evolved resistance to bensulfuron-methyl. Five populations from Hunan Province (HN-2, HN-3, HN-5, HN-9, HN-10) possessing the Trp-574-Leu mutation had high-level resistance (ranging from 169- to >1,309-fold) based on GR₅₀ ratios. The resistant populations from Guangxi Province had a lower level of resistance to bensulfuron-methyl due to a Pro-197-Ser mutation. The Asp-376-Glu mutation was only identified in the HN-4 population. In addition, the GX-3 population from the PTR systems was resistant to bensulfuron-methyl without ALS gene mutations, indicating non–target site resistance (NTSR). Although some resistant populations of both regions exhibited cross-resistance to multiple ALS-inhibiting herbicides, including pyrazosulfuron-ethyl, bispyribac-sodium, penoxsulam, and imazapic, sensitivity was also detected to the auxin herbicide MCPA and the photosystem II–inhibiting herbicides bentazone and propanil. These results indicate that cultivation practices affect resistance evolution in C. difformis. DSR systems exert high selection pressure by selecting the Trp-574-Leu mutation, resulting in high-level resistance. In contrast, a mutation at Pro-197 plus NTSR likely plays a significant role in ALS resistance in the PTR systems.