Legacy of Recurrent Gene Flow and Evidence of Independent Evolution of ALS Resistance Mutations in Weedy Rice

ABSTRACT

Weedy rice ( Oryza spp.) provides a unique model for studying adaptive evolution because herbicide resistance can arise through both crop‐to‐weed gene flow and independent mutational events. In Brazil, nearly two decades of cultivation of imidazolinone (IMI)‐resistant rice have imposed intense selection for acetolactate synthase (ALS) inhibitors resistance. This study investigated how historical gene flow and independent evolution have shaped the contemporary landscape of ALS resistance mutations in Brazilian weedy rice. Specifically, we characterized the diversity and distribution of ALS resistance mutations and developed rapid allele‐specific molecular markers for major resistance alleles. A total of 200 weedy rice accessions collected from the major rice‐growing region of Brazil were screened for IMI resistance and genotyped using newly developed SNAP (single nucleotide amplified polymorphism) qPCR markers targeting the IMI rice‐derived mutations Ala122Thr, Ser653Asn, and Gly654Glu. Samples negative for these mutations were sequenced, and dose‐response assays were conducted with imazethapyr, penoxsulam, nicosulfuron, and cloransulam‐methyl. Ninety‐three percent of the accessions were resistant to IMI herbicides. The ALS mutation Gly654Glu, present in rice cultivars used until 2006, was found in 15.5% of weedy rice resistant accessions, Ala122Thr became the dominant mutation (54.5%), whereas Ser653Asn occurred at an intermediate frequency (16.0%), indicating a temporal shift in the genetic basis of ALS resistance. Sequencing further identified the ALS substitutions Ala205Val, Trp574Leu, and Ser653Thr, which were not associated with commercial IMI‐resistant rice cultivars, indicating ongoing independent evolution of target‐site resistance. Trp574Leu conferred broad cross‐resistance to all ALS‐inhibiting herbicides evaluated, whereas the remaining mutations displayed mutation‐specific resistance spectra. The SNAP markers showed high specificity and sensitivity, enabling reliable high‐throughput detection of resistance alleles. Together, our findings demonstrate that the contemporary ALS resistance landscape in Brazilian weedy rice has been shaped mainly by repeated crop‐to‐weed gene flow, but also ongoing independent mutational events that continue to diversify resistance under herbicide selection.