Detection of Two Common ACCase Mutations Associated with High Levels of Fenoxaprop-P-Ethyl Resistance in Shortawn Foxtail (Alopecurus aequalis) Using Loop-Mediated Isothermal Amplification

Abstract

The resistance to fenoxaprop-P-ethyl, a herbicide that inhibits acetyl-CoA carboxylase (ACCase), has emerged in shortawn foxtail (Alopecurus aequalis Sobol.) since the 1990s, presenting a considerable challenge to wheat (Triticum aestivum L.) production in China. One of the primary mechanisms responsible for this high-level resistance is the presence of mutations at codons 1781, 2041, and 2078 in the ACCase gene. However, the conventional methods used to detect these mutations, such as polymerase chain reaction (PCR) and gene sequencing, are time-consuming and labor-intensive. To address this issue and enable the prompt and effective detection of these common ACCase mutations in A. aequalis, a loop-mediated isothermal amplification (LAMP) strategy was developed. The LAMP assay specifically targets the Ile-1781-Leu and Asp-2078-Gly mutations within the ACCase gene of A. aequalis. Through the optimization of primers, systems, and conditions, the LAMP assay enables rapid differentiation between wild-type individuals and mutants of A. aequalis carrying either of these two mutations. By including SYBR Green I dye in the final reaction mixtures, the target mutation can be visually detected through a noticeable color change that can be observed with the naked eye. It is noteworthy that the sensitivity of the LAMP assay was approximately 10⁴-fold greater than that of conventional PCR methods. Additionally, a derived cleaved amplified polymorphic sequence (dCAPS) assay was established for each mutation to distinguish between homozygous and heterozygous mutants. Overall, the developed LAMP assay could efficiently detect the Ile-1781-Leu and Asp-2078-Gly mutations in the ACCase gene of A. aequalis, offering significant advantages for the monitoring and management of fenoxaprop-P-ethyl resistance.