Validation of reference genes across populations of Aphis glycines Matsumura for RT-qPCR analysis of gene expression related to pyrethroid detoxification

Metabolic detoxification is a common mechanism of insecticide resistance in insects, in which detoxifying enzyme genes are overexpressed. Aphis glycines is one of the major soybean pests in the USA and it has developed resistance to pyrethroid insecticides after almost two decades of its management mostly using pyrethroids. To date there are no validated reference genes to normalize gene expression for A. glycines in the context of pyrethroid resistance. In this study, we compiled a list of 68 reference genes frequently used in gene expression analysis in insects, mostly aphids, and performed a three-phase screening. In the first level of screening, we narrowed the list down to 30 candidate reference genes based on amplification efficiency in one population of A. glycines. In the second screening, the list was further reduced to 14 genes based on the percentage of missing values analyzed with LingReg PCR across nine A. glycines populations with different levels of pyrethroid resistance from Minnesota and Iowa, USA. The stability of the gene expression was evaluated using NormFinder and a generalization of BestKeeper. The third level of screening, which was a final validation of the stability of the genes, resulted in six genes [actin, RPL9 (Ribosomal protein L9), RPS9 (Ribosomal protein S9), AK (Arginine kinase), RNAPol2 (RNA polymerase II) and RPL17 (Ribosomal protein L17)] identified as the most suitable reference genes. Our findings will support studies related to insecticide resistance in A. glycines.