Validation of Reference Genes Across Populations of Aphis glycines (Hemiptera: Aphididae) for RT-qPCR Analysis of Gene Expression Related to Pyrethroid Detoxification

Metabolic detoxification is a common mechanism of insecticide resistance, in which detoxifying enzyme genes are overexpressed. Aphis glycines Matsumura (Hemiptera: Aphididae) is one of the major soybean pests in the United States and has developed resistance to pyrethroid insecticides after almost two decades of use. To date, there are no validated reference genes to normalize expression of detoxification genes for pyrethroid resistance in A. glycines. From a literature review, a list was compiled of genes from 36 gene families (68 sequences) frequently used as reference genes in gene expression analysis in Hemiptera. Exon-exon junction primers were designed for the best alignment matches to a draft A. glycines genome and were assayed in a three-phase screening. The first screen eliminated nonamplifying primers. The second screen used nine A. glycines populations varying in resistance to pyrethroids and eliminated primers with inconsistent amplification or low amplification efficiency, and quantitatively assessed the stability of expression in the 14 remaining candidates using NormFinder and a generalization of BestKeeper. The third screen quantitatively validated these results on the best candidates. Six genes were identified with the greatest stability across technical and biological replication and the nine populations. The genes identified as the most suitable reference genes for the study of detoxifying enzymes related to pyrethroid resistance in soybean aphid were: Actin, RPL9 (ribosomal protein L9), RPS9 (ribosomal protein S9), AK (arginine kinase), RNAPol2 (RNA polymerase II), and RPL17 (ribosomal protein L17). Our findings will support studies related to insecticide resistance in A. glycines.