Selection of Reference Genes for Normalization of qRT‒PCR Analysis in the Soybean Aphid Aphis glycines Matsumura (Hemiptera: Aphididae)

The soybean aphid Aphis glycines Matsumura is a predominant insect pest in Asia and North America and causes great losses to soybean. The release of genome data for A. glycines will facilitate gene function research in the future. However, suitable reference genes for A. glycines under various experimental conditions are scarce. To search for appropriate reference genes for A. glycines, nine candidate reference genes, including Act, α-Tub, β-Tub, RPS12, RPS18, RPL5, RPL27, EF1α, and Fer, were tested under six experimental conditions to evaluate their suitability for use in the normalization of qRT‒PCR data. Results showed that EF1α and RPS12 were optimal for the developmental stages of A. glycines, RPS18 and RPS12 were appropriate for wing dimorphism, β-Tub and RPS18 were suitable for different tissues and RPL5, and α-Tub could be used for normalization at different temperatures. β-Tub and EF1α could be proposed as reference genes for insecticide treatment, and RPL5 and RPS12 were found to be the most stable reference genes in different photoperiods. The results provide appropriate reference genes for analyzing gene expression in A. glycines and contribute to future research on the molecular physiology and biochemistry of A. glycines.