Abstract: 【Aim】 This study aims to select the stably expressed reference genes for expression analysis of microRNAs (miRNAs) in the cereal aphid, Sitobion avenae, under specific conditions. 【Methods】 According to the Illumina sequencing results of S. avenae miRNAs, 10 candidate reference genes including miR-10-3p, miR-993, miR-276, miR-275, miR-252a, miR-1, miR-375, pc-15, pc-73 and one normal reference gene U6 were screened, and their relative expression levels in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids and in wingless aphids of S. avenae exposed to four insecticides (95.1% imidacloprid technical material, 97.8% thiacloprid technical material, 95% avermectins technical material and 40% omethoate emulsifiable concentrate) were detected by using qRT-PCR. The expression stability of the 10 candidate reference genes was evaluated by GeNorm, NormFinder, ΔCt method, BestKeeper, and RefFinder. 【Results】 The qRT-PCR results showed that the highly expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-276, miR-276, miR-10-3p, and miR-10-3p, respectively. Integrating the analysis results of five approaches, more stably expressed reference genes under the above four conditions were miR-252a, miR-993, miR-275, and miR-993, respectively. The optimal number of reference genes was two under different conditions by GeNorm. Based on the comprehensive ranking of RefFinder, the optimal combinations of stably expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. 【Conclusion】 The optimal combinations of reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to different insecticides are miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. The results provide a basis of reference gene selection for quantitative expression analysis of miRNA genes in S. avenae.

Key words: Sitobion avenae, reference gene, miRNA, expression stability, qRT-PCR