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Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified  ClTUA  and  ClACT ,  ClEF1α  and  ClACT , and  ClCAC  and  ClTUA  as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified  ClYLS8 ,  ClUBCP , and  ClCAC  as the best single reference genes under the above experimental conditions, respectively.  ClYLS8  and  ClPP2A  were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene  18SrRNA  was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes.  ClCAT1 and  ClCAT2  were induced and upregulated in the first 24 h, whereas  ClCAT3  was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when  18SrRNA  was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon.

Identification of Suitable Reference Genes for Gene Expression Normalization in qRT-PCR Analysis in Watermelon