Abstract
Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.
Highlights
Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae), commonly known as spotted stem borer (SSB), is an invasive and polyphagous pest that hampers maize production worldwide[1,2]
Of the six candidate reference genes analysed in Phenacoccus solenopsis, β-Tubulin was found to be the most stable reference gene in most tested conditions17. 16S, RPS18 and RPL13 were recommended for normalization of RT-qPCR data in L. erysimi[18]
We evaluated the stability of seven reference genes, 18S (18S ribosomal RNA), V-ATPase, Actin(beta-Actin), ELF (Elongation factor 1 alpha), RPL32, Tubulin and HSP70 (Heat shock protein 70) (Table 1), representing separate gene families and functional classes and using five software algorithms, the comparative deltaCt method[19], BestKeeper[20], geNorm[21], NormFinder[22], and online platform-RefFinder[23]
Summary
Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae), commonly known as spotted stem borer (SSB), is an invasive and polyphagous pest that hampers maize production worldwide[1,2]. Most RNAi studies on agricultural pests failed to validate the best reference gene for effective evaluation of silencing effects. There is insufficient genomic information in C. partellus to identify a reference gene that has stable expression under the translational inhibitory effect of dsRNA across various experimental conditions and developmental stages in Chilo partellus. To increase the credibility of RT-qPCR data interpretation, effective normalization is required to nullify background variation and biases that might influence the expression of transcript[10,11] due to differential expression of reference genes under specific conditions[12,13]. 16S, RPS18 and RPL13 were recommended for normalization of RT-qPCR data in L. erysimi[18] Such variation in insect reference genes across different experimental necessitates reference gene validation depending on the experiment. Our outcome will be useful in future studies for gene expression analysis on RNAi-mediated control against this notorious pest ravaging many parts of the world
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