Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Shipeng Han,Qiuju Qin,Yayuan Zhou,Da Wang,Yunzhuan He

doi:10.3390/insects12100902

Abstract

Simple SummaryThe fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is an extremely important omnivorous agricultural pest, it poses a severe threat to food security and agricultural production. Quantitative real-time PCR (qRT-PCR) is an important molecular technology widely used for expression profile analyses of various target genes. It is essential to use reference genes as the benchmark to eliminate various errors and normalize the qRT-PCR analysis. In our study, 10 reference genes were evaluated under six experimental conditions, including developmental stages, tissues, mating status, hormones, diets, and temperatures. Finally, the expression profile of the target gene SfrOBP1 in various tissues of S. frugiperda was evaluated to verify the accuracy of the results. This study will provide a preliminary evaluation of reference genes of S. frugiperda, which can be beneficial to the further research of functional gene expression.As an accurate and convenient technique, the qRT-PCR is always used in the quantitative expression analysis of functional genes. Normalization of the data relies on stable reference genes. The fall armyworm Spodoptera frugiperda (J. E. Smith) is an important invasive and migratory pest that seriously threatens corn production around the world. In this paper, we selected 10 candidate reference genes (18S, AK, RPL10, RPS24, 28S, SOD, ATP, GAPDH, ACT, and a-TUB) and determined their expression levels under different conditions (different developmental stages, various tissues, mating status, hormones, diets, and temperatures). Subsequently, the stability of reference genes was evaluated by four algorithms (Delta Ct method, geNorm, NormFinder, BestKeeper). The optimal combination of reference genes for each treatment was obtained by geNorm. Finally, the comprehensive ranks were determined by the online tool RefFinder. Results showed that the most stable reference genes were SOD, RPL10, and RPS24 for developmental stages, α-TUB, RPL10, and ATP for different tissues, AK, RPL10, and 18S for mating status, 18S and AK under hormone treatment, 18S, RPL10, and SOD under diet treatment, RPL10, 18S, and RPS24 under temperature treatment. This study confirmed recent data on a few reference genes and provided an evaluation of a number of additional reference genes of S. frugiperda under various conditions.

Highlights

The expression stability of ATP was highest in BestKeeper
The Quantitative real-time PCR (qRT-PCR) is a reliable technique in gene expression analysis, with high sensitivity and specificity, and the qRT-PCR data must be normalized by suitable reference genes to avoid expression differences among samples [31]
The results showed that SOD for different developmental stages, α-TUB for various tissues, AK for mating treatment, 18S for hormone treatment, ATP for diets treatment, RPL10 for temperature treatment were the most stable reference genes of S. frugiperda

Summary

Introduction

E. Smith) (Lepidoptera: Noctuidae) is indigenous to the tropical and subtropical regions of the American continent, with high reproductive capacity, a wide range of hosts, and strong migration ability [1]. Smith) (Lepidoptera: Noctuidae) is indigenous to the tropical and subtropical regions of the American continent, with high reproductive capacity, a wide range of hosts, and strong migration ability [1] This pest seriously threatens corn production and food security around the world [2,3,4]. The S. frugiperda has invaded 46 countries or regions of Africa and nine countries of

Results

Discussion

Conclusion