Abstract
Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.
Highlights
Watermelon (Citrullus lanatus) is a popular and economically important horticultural crop in terms of production and consumption
Each reference gene amplified a specific product on cDNA templates, but no products were detected on genomic DNA (gDNA) templates except for ClACT and Cl18SrRNA, demonstrating the success of primer design (Fig 1)
No signals were detected in the controls without templates in quantitative reverse-transcription PCR (qRT-PCR) reactions
Summary
Watermelon (Citrullus lanatus) is a popular and economically important horticultural crop in terms of production and consumption. Watermelon fruits undergo various physiological and biochemical changes, resulting in diverse sizes, colors, shapes, sweetness, textures, and aromas [1]. Watermelon is gaining popularity as a model plant for studying non-climacteric fruits. Studies have focused on the unique metabolic and regulatory networks of watermelon fruit to improve the crucial nutritional attributes of this fruit [2].
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