Loop-mediated isothermal amplification for detecting the Ile-2041-Asn mutation in fenoxaprop-P-ethyl-resistant Alopecurus aequalis.

Background: Shortawn foxtail (Alopecurus aequalis Sobol.), a competitive grass weed severely infesting overwintering crops worldwide, has evolved resistance to the highly efficient acetyl-CoA carboxylase (ACCase)-inhibiting herbicide fenoxaprop-P-ethyl. The Ile-to-Asn substitution at codon position 2041 of ACCase is a dominant resistance mutation that has been associated with fenoxaprop-P-ethyl resistance in A. aequalis. However, its detection based on conventional methods such as polymerase chain reaction (PCR) and gene sequencing is rather labor- and time-consuming.

Results: In order to facilitate its detection in field populations of A. aequalis, a simple and efficient method with high sensitivity to the Ile-2041-Asn mutation was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed to target a 244-bp fragment of ACCase comprising codon position 2041. Using the special primers and genomic DNA of A. aequalis, the concentrations of reaction components, temperature and time each were optimized. The LAMP reaction for the detection of the Ile-2041-Asn mutation was processed at 65 °C for 45 min followed by 80 °C for 10 min to stop the reaction. The LAMP method developed was 1000-fold more sensitive than the conventional PCR method, and the detection was also practicable when using crude DNA of A. aequalis as a template.

Conclusion: The low cost, simplicity and high sensitivity of the developed LAMP assay make the detection of the Ile-2041-Asn mutation easier and quicker, which may contribute to the monitoring and management of resistance development to fenoxaprop-P-ethyl in A. aequalis. © 2022 Society of Chemical Industry.