Evaluation of Watermelon Reference Genes

A total of 15 candidate reference genes were evaluated. These genes were chosen based on their previous use in watermelon or their validation as best reference genes in other crops, including 18S ribosomal RNA (18SrRNA), β-actin (ACT), clathrin adaptor complex subunit (CAC), elongation factor 1-α (EF1α), glyceraldehy-3-phosphate-dehydrogenase (GAPDH), NADP-isocitrate dehydrogenase (IDH), leunig (LUG), protein phosphatase 2A regulatory subunit A (PP2A), polypyrimidine tract-binding protein 1 (PTB), ribosomal protein S (RPS2), SAND family protein (SAND), α-tubulin (TUA), ubiquitin-conjugating enzyme E2 (UBC2), ubiquitin carrier protein (UBCP), and yellow-leaf-specific proein8 (YLS8).
For each candidate reference gene, blastn was carried out in the Cucurbit Genomics Database (http://www.icugi.org) against watermelon coding DNA sequences (CDS) (v1) using Arabidopsis homolog as a query. The CDS of the best hit was retrieved and uploaded to Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) for primer design. The product size was set at the range of 80 bp to 150 bp. The forward and reverse primers were intentionally targeted on the adjoining exons, which were separated by an intron. The generated primer pair for each gene was then aligned against all watermelon CDS to confirm its specificity in silico. The specificity of the PCR amplification product for each primer pair was further determined by electrophoresis in 2% agarose gel and melting curve analysis. Finally, the watermelon species name abbreviation of ‘Cl’ was added as a prefix to the specificity-validated gene to specify the watermelon orthologous gene. For more comparable results, the primer pair of 18SrRNA, which was previously published, was used in this study [2] (link). Data on the selected reference genes and their amplification characters are listed in Table 1.