Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Real-time quantitative PCR (qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha (EF1 alpha), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), ribosomal protein L13 (RPL13), alpha-tubulin (alpha -TUB), and beta-1-tubulin (beta -1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by Delta Ct, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation (V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and beta -1-TUB for larval tissue samples, EF2 and EF1 alpha for the larval samples treated with different temperatures, RPL3 and EF1 alpha for the larval samples under starvation stress, and RPL13 and EF1 alpha for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.

Automated summary

This study evaluated the stability of eight candidate reference genes in the fall armyworm and identified the most stable reference gene combinations for different experimental conditions, laying the foundation for the normalization of qPCR analyses in this pest.