Reliable RT-qPCR Normalization in Polypogon fugax: Reference Gene Selection for Multi-Stress Conditions and ACCase Expression Analysis in Herbicide Resistance

Asia minor bluegrass (Polypogon fugax), a widespread Poaceae weed, exhibits broad tolerance to abiotic stresses. Validated reference genes (RGs) for reliable RT-qPCR normalization in this ecologically and agriculturally significant species remain unidentified. This study identified eight candidate RGs using transcriptome data from seedling tissues. We assessed the expression stability of these eight RGs across various abiotic stresses and developmental stages using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. A comprehensive stability ranking was generated using RefFinder, with validation performed using the target genesCOR413andP5CS. Results identifiedEIF4AandTUBas the optimal RG combination for normalizing gene expression during heat stress, cold stress, and growth stages.EIF4AandACTwere most stable under drought stress,EIF4Aand28Sunder salt stress, andEIF4AandEF-1 under cadmium (Cd) stress. Furthermore,EIF4AandUBQdemonstrated optimal stability under herbicide stress. Additionally, application of validated RGs revealed higher acetyl-CoA carboxylase gene (ACCase) expression in one herbicide-resistant population, suggesting target-site gene overexpression contributes to resistance. This work presents the first systematic evaluation of RGs inP. fugax. The identified stable RGs provide essential tools for future gene expression studies on growth and abiotic stress responses in this species, facilitating deeper insights into the molecular basis of its weediness and adaptability.