Selection and Evaluation of Reference Genes for Quantitative Real-Time PCR in Tomato (Solanum lycopersicum L.) Inoculated with Oidium neolycopersici

Abstract

In order to screen out the most stable reference genes in tomatoes under powdery mildew (PM) stress and study the expression of related genes in the interaction between tomato and PM more accurately, this study will provide a calibration basis for the expression of related functional genes. In this study, the expression stabilities of eight tomato candidate reference genes of EF1α, L33, Act, Ubi, GAPDH, UK, CAC and TIP41 in susceptible tomato and resistant tomatoes under PM stress were ranked using four different computation programs, including geNorm, Normfinder, BestKeeper and the comparative ∆CT method. Then RefFinder was used to analyze the ranking results of four kinds of software comprehensively. Finally, the selected reference genes were validated by the target gene SlMLO1. The results of geNorm showed that the normalization of qRT-PCR using two reference genes could meet the requirements. The comprehensive analysis of RefFinder showed that the most stable reference genes were Act and EF1α for both tomato varieties. The combination of Act and GAPDH was most stable in susceptible tomato ‘MM’. The combination of Act and EF1α was most stable in resistant tomato ‘62579′. Generally, the Act was the most stable reference gene in the two tomato varieties under PM stress. This study will lay a foundation for the related functional gene expression research in tomatoes under PM stress.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

“…For this reason, RefFinder stands as an interesting alternative, instead of using these algorithms individually, since it uses the individual results obtained by each algorithm to perform a reanalysis and generate an overall classification of the most stable genes 66 . The RefFinder tool has been widely used in studies aimed at determining the best reference genes 51 , 54 , 56 , 74 .…”

“…Therefore, their appropriate selection, as performed in this research, is a fundamental preliminary step in gene expression studies, since the use of inappropriate reference genes can significantly compromise the accuracy and interpretation of the results 33,50 . In this sense, several studies have been carried out, in different organisms and experimental conditions, with the aim of selecting and validating the best reference genes for RT-qPCR gene expression analyses [51][52][53][54] .…”

“…This contributes to the reliability of the analyses performed in this study and reinforces the importance of carefully evaluating gene expression levels when aiming to select the most appropriate reference genes for normalizing RT-qPCR gene expression data. Furthermore, this analysis has been widely explored in several studies with similar objectives 51,54,[56][57][58] .…”

“…The algorithms used in this study, geNorm 59 , NormFinder 62 , BestKeeper 63 , Delta-Ct 64 , and RefFinder 65,66 have been commonly used to evaluate the stability of reference genes in plants 19,25,54 , animals [67][68][69] and microorganisms [70][71][72] . They are used to calculate the stability of gene expression using different mathematical models.…”

Efficacy of 2,4-Di-tert-butylphenol in Reducing Ralstonia solanacearum Virulence: Insights into the Underlying Mechanisms

“…One microgram of RNA was used to obtain cDNA, and qPCR was performed as previously described. The glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) gene of tomato was used as the internal reference, 23 and the specific amplification of R. solanacearum was carried out using the RSF gene ( Table 1 ). Custom primers for the evaluated pathogenic genes were designed by using the Primer5 software.…”