Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Abstract

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR

“…We found that no standard reference gene is suitable for RT–qPCR analysis. Reference genes, such as actin, tubulin, tef1, NADP, and ubiquitin, are widely used in different species 18 , 20 , 22 – 24 . However, appropriate reference genes are not uniform across diverse species, and even in different treatments or tissues of the same species 18 – 23 .…”

“…Actin, GAPDH, tubulin, and ubiquitin are used commonly in analysing gene expression levels in plants 23 – 26 . However, there are no standards to use as reference genes in various species or tissues 24 . The best reference genes were generally confirmed by analytical tools, such as GeNorm, NormFinder, and BestKeeper 25 .…”

“…However, the scores for reference genes evaluated sing these three methods are often not uniform. Therefore, researchers have selected genes that have high scores in GeNorm and NormFinder or used a combination of the three methods 24 . As a result, the best reference gene remains to be identified.…”

Evidence-based insecticide resistance in South American tomato leaf miner,Phthorimaea absoluta(Meyrick) under laboratory selection

“…Expression of the identified CYP genes was performed using RT-qPCR (Real-Time Quantitative Reverse Transcription PCR) with their gene-specific primers (Supplementary table 2) with the internal reference gene, RPS-13 (Yang et al ., 2021). The cDNA from G1 and G20 population was prepared and diluted at 10 ng μl −1 before setting the reaction.…”

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae)

“…Ultimately, the online tool RefFinder (https://www.heartcure.com.au/reffinder/, accessed on 8 October 2023) was employed to determine the weighted geometric means in the form of stability values derived from the aforementioned algorithms. This allowed for the establishment of a consensus ranking regarding the stability of the reference genes [29][30][31]. Moreover, the GeNorm algorithm was used to determine the most suitable quantity of reference genes by evaluating pairwise variance values (V).…”