AUTHOR=Fu Haiyan , Huang Tubiao , Yin Cheng , Xu Zhenhua , Li Chao , Liu Chunguang , Wu Tong , Song Fuqiang , Feng Fujuan , Yang Fengshan 

TITLE=Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

JOURNAL=Frontiers in Physiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.818210

DOI=10.3389/fphys.2021.818210

ISSN=1664-042X

ABSTRACT=<p><italic>Bradysia odoriphaga</italic> (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling <italic>B. odoriphaga</italic>. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in <italic>B. odoriphaga</italic>, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [<italic>elongation factor 1α (EF-1α)</italic>, <italic>actin2 (ACT)</italic>, <italic>elongation factor 2α (EF-2α)</italic>, <italic>glucose-6-phosphate dehydrogenase (G6PDH)</italic>, <italic>glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</italic>, <italic>ribosomal protein L10 (RPL10)</italic>, <italic>ribosomal protein S3 (RPS3)</italic>, <italic>ubiquitin-conjugating enzyme (UBC)</italic>, and <italic>α-tubulin (TUB</italic>)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and Δ<italic>Ct</italic>) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were <italic>RPL10</italic> and <italic>ACT</italic> across phoxim; <italic>ACT</italic> and <italic>TUB</italic> across chlorpyrifos and chlorfluazuron; <italic>EF1</italic>α and <italic>TUB</italic> across imidacloprid; <italic>EF1</italic>α and <italic>EF2</italic>α across developmental stages; <italic>RPL10</italic> and <italic>TUB</italic> across larvae; <italic>EF1</italic>α and <italic>ACT</italic> across tissues, and <italic>ACT</italic> and <italic>G6PDH</italic> across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on <italic>B. odoriphaga</italic> gene function under insecticides stress.</p>