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

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

doi:10.3389/fphys.2021.818210

Haiyan Fu, Zhenhua Xu + Show 8 more

Open Access

https://doi.org/10.3389/fphys.2021.818210

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Abstract

Bradysia odoriphaga (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 B. odoriphaga. 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 B. odoriphaga, 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 [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] 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 ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

Highlights

Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) is a serious soil pest in China that feeds on 7 plant families and more than 30 plant species, including Chinese chive (Liliaceae), onion (Liliaceae), Chinese cabbage (Cruciferae), lettuce (Asteraceae), and so on
The specific amplification of all primer pairs of candidate reference genes was confirmed with regular PCR and RT-qPCR
Earlier studies on reference genes evaluation and validation in insects under insecticide stress reported that the expression of reference genes varies under different insecticide stress even if they belong to the same group of insecticides (Liang et al, 2014)

Summary

Introduction

Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) is a serious soil pest in China that feeds on 7 plant families and more than 30 plant species, including Chinese chive (Liliaceae), onion (Liliaceae), Chinese cabbage (Cruciferae), lettuce (Asteraceae), and so on B. odoriphaga larvae usually gather in the roots, bulbs, and even in immature stems of Chinese chives, making the pest hard to control and allowing it to cause significant production losses of Chinese chives (Zhang et al, 2013; Chen et al, 2018). The control efforts against B. odoriphaga still largely rely on the application of chemical insecticides, such as organophosphate (OP) and neonicotinoid insecticides (Chen et al, 2017). Further studies on the mechanism of insecticide resistance are required to clarify the genes directly involved in resistance and regulatory mechanisms associated with those genes

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