Analysis of Candidate Gene Expression Levels

The Cla009408, Cla006737, Cla006738, Cla001244, Cla006625, Cla009378, Cla009382, Cla007521, and Cla009410 expression patterns were examined by quantitative real-time (qRT)-PCR. The G17AB and “Zhongliu” plants were grown for about 60 days after sowing. Three replicates of the anther samples were collected, each comprising 10 anthers from individual plants. Total RNA was extracted and gene expression was analyzed as previously described (Dong et al., 2018). Briefly, total RNA was isolated from each sample using the TRIzol reagent (Invitrogen, Carlsbad, CA, United States). qRT-PCR was completed using TB Green^® Premix Ex Taq^TM II (Tli RNaseH Plus) and the Roche LightCycler 480 II instrument according to the manufacturer’s instructions. The PCR program was as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s. The ClYLS8 gene was used as an internal reference control. Primers were designed based on the C. lanatus 97,103 genome sequence using the Primer5 program. Details regarding the qRT-PCR primers used to analyze candidate gene expression are listed in Table 1.

Sequence details for the qRT-PCR primers used to analyze watermelon candidate gene expression.