Abstract
Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.
Highlights
Tomato (Solanum lycopersicum) is an economically important horticultural crop in terms of production, flavor, and nutritional value of fruits
Tomato (S. lycopersicum L.) genome database in the SGN and transcriptome data derived from the TFGD were used for Primer sequence (5′–3′)
According to our extensive evaluation, five identified reference genes (RGs)-SlFRG05, SlFRG12, SlFRG16, SlFRG27, and SlFRG37 could be recommended for normalization of quantitative real-time PCR (qPCR) experiments in tomato fruits
Summary
Tomato (Solanum lycopersicum) is an economically important horticultural crop in terms of production, flavor, and nutritional value of fruits. During the course of development and ripening, tomato fruits undergo a number of physiological and biochemical processes that bring forth the overall changes in fruit size, color, texture, and aroma (Klee and Giovannoni, 2011; Ruiz-May and Rose, 2012). A number of studies had been carried out to improve agronomic traits of tomato fruits, including size, pigment content, and flavor substances focusing on the metabolic and regulatory networks (Klee and Giovannoni, 2011; Ruiz-May and Rose, 2012; Tieman et al, 2017). RGs for Tomato Fruit Development resources and bioinformatics tools (e.g., Genome-wide association study, GWAS) have enabled rapid elucidation of the complicated biological processes that occur during fruit development. Quantitative real-time PCR (qPCR) is considered as one of the most efficient tools for the measurement of transcript abundance of a gene due to its high accuracy, sensitivity, and reproducibility (Ginzinger, 2002; Bustin and Nolan, 2004; Gachon et al, 2004; Bustin et al, 2005)
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