Screening potential reference genes for reverse transcription‐quantitative polymerase chain reaction normalization in Atrijuglans hetaohei (Lepidoptera: Gelechioidea)

Atrijuglans hetaohei Yang (Lepidoptera: Gelechioidea), is one of the major pests that seriously damage the walnut fruits. Although the morphology and physiology of A. hetaohei have been widely studied, suitable reference genes for normalizing target gene expression have not been identified. In this study, the expressions of eight candidate reference genes including GAPDH, β‐Tubulin, TATA‐binding protein (TBP), Histone, Ubiquitin‐conjugating enzyme 9 (UBC9), arginine kinase (AK), 28S and elongation factor 1 alpha (EF1α) in different developmental stages and various larval tissues of A. hetaohei were determined using reverse transcription‐quantitative polymerase chain reaction. Moreover, the expression stability of the selected reference genes was evaluated by ΔCt method, BestKeeper, NormFinder, geNorm and ReFinder, a comprehensive software platform. These results demonstrated that the best reference genes were GAPDH and 28S at different developmental stages of A. hetaohei; and the optimum references in various larval tissues of A. hetaohei were 28S, Histone and TBP. Our study should be useful to analyze the expression profiles of target genes and form a solid foundation for future research on the understanding of the genes' biological functions in A. hetaohei.