Quantification of Resistance Gene Expression

The transcript levels of non-receptor genes were quantified by real-time qPCR performed in a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Gene-specific primers for PxAPN5, PxAPN6, and PxABCC1 were used (Additional file 1: Table S1) in qPCR reactions with 2.5×SYBR Green MasterMix Kit (TIANGEN) as described in detail elsewhere [22 (link), 27 (link)]. The qPCR program consisted of an original denaturation step for 6 min at 95 °C, subsequently, 40 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 53 °C for PxAPN6, 55 °C for PxABCC1 and PxAPN5, followed by an extension for 35 s at 72 °C. Relative quantification was calculated by utilizing the 2^−ΔΔCt method and standardized to the ribosomal protein L32 gene (GenBank accession no. AB180441). Three biological repetitions and four technical replicates were performed for each sample.