Background Benzobicyclon (BBC) is a β-triketone herbicide (bTH) used in rice paddy fields. It has the advantages of high efficiency, low toxicity, high crop safety, and good environmental compatibility, and shows efficacy against paddy weeds resistant to other types of herbicides. However, because BBC is not safe for some important indica rice varieties, it is currently only registered and applied in japonica rice areas. Results In this study, by analyzing haplotypes of the bTHs broad-spectrum

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... esistance gene HIS1 and phenotypes for BBC in 493 major indica rice accessions in China, we identified a novel non-functional allelic variant of HIS1 besides the previously reported 28-bp deletion. Through detection with markers specific to the two non-functional mutations, it was clear that 25.4% of indica conventional varieties, 59.9% of restorers and 15.9% of sterile lines were susceptible to BBC. In addition, due to natural allelic variations of the HIS1 gene in sterile and restorer lines, some two-line hybrid sterile lines were sensitive to bTHs, and the corresponding restorers were resistant. We showed the potential of effectively using bTHs to solve the problem of two-line hybrid rice seed purity due to self-crossing of sterile lines during hybrid rice seed production. Finally, allelic variations of the HIS1 gene may also play an important role in the mechanized seed production of hybrid rice. Conclusions Our findings provide guidance for the application of BBC in indica rice areas, and provide a non-transgenic approach to solve the problem of seed purity of two-line hybrid rice.