Acetolactate Synthase Overexpression in Mesosulfuron-Methyl-Resistant Shortawn Foxtail (Alopecurus aequalis Sobol.): Reference Gene Selection and Herbicide Target Gene Expression Analysis

Severe infestations of shortawn foxtail (Alopecurus aequalis Sobol.), a noxious weed in wheat and canola cropping systems in China, remain standing even after the application of the herbicides, fenoxaprop-P-ethyl and mesosulfuron-methyl. Analysis of gene expression in weed plants subjected to herbicide treatment is a key step toward more mechanistic studies. Since such an analysis often involves quantitative real-time PCR (qRT-PCR), endogenous reference genes with stable expression are required. Herein, we identified specific gene sets, suitable as references for qRT-PCR data normalization in A. aequalis plants under different experimental conditions, using geNorm, NormFinder, BestKeeper, and RefFinder software. Additionally, the reliability of reference genes was verified by analyzing the expression of genes encoding two major herbicide target enzymes: acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS). Furthermore, functional ALS gene amplification was likely present in resistant plants, although it may make no contribution to the resistant phenotypes.