Molecular characterization and detection of a spontaneous mutation conferring imidazolinone resistance in rapeseed and its application in hybrid rapeseed production

Abstract

Imidazolinone herbicide (IMI)-resistant varieties of many crops, induced by mutations to acetohydroxyacid synthase (AHAS) genes, are planted worldwide. However, in the case of rapeseed, which is a common source of edible oil for nearly one-half of China’s population, no IMI resistance has been reported for any of the varieties currently cultivated. We have developed an imazethapyr-resistant rapeseed (M9) derived from a naturally occurring mutant plant. The goals of this study were to determine the biochemical and molecular bases of herbicide resistance in M9, to develop molecular markers for the detection of herbicide-resistant genes, and to utilize herbicide-resistant traits to enhance seed purity in hybrid rapeseed production. An in vitro AHAS activity assay indicated that the AHAS enzyme from M9 conferred a specific resistance to IMIs. Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 653 (AGT) to asparagine (AAT) at the herbicide-binding site of the rapeseed BnAHAS1 gene. This substitution mutation (Ser653Asp) did not change the transcription levels of BnAHAS1 in M9 compared with the wild type. An allele-specific PCR marker for the BnAHAS1 mutant sequence was developed and cosegregated with IMI resistance in the F2, BC1, and BC2 populations. Finally, the CMS restorer line 10M169 was developed to show the resistance of M9, and F1 seeds of different purity were generated from 10M169 and Ning A7 under different pollination conditions. The increases in seed purity under natural hybridization and hybridization in tents reached 13.41 and 16.41 %, respectively, after IMI treatment, suggesting that the herbicide-resistant trait can be utilized for the efficient elimination of false hybrids in hybrid rapeseed production, leading to increases in yield of up to 322 and 394 kg ha−1, respectively. The molecular mechanism and molecular marker of herbicide resistance described here provide the basis for the release of IMI-resistant rapeseed cultivars.