Plant total rna purification kit

To validate the SlNAC2 transcript level in wild-type, pBI121 plants, and overexpression transgenic lines of SlNAC2, the Plant Total RNA Purification Kit (Tiangen, Beijing, China) was used to extract total RNA from 7-day-old seedlings. Then, using the reverse transcription kit (TransGen Biotech, Beijing, China), 1.5 ug RNA was reverse-transcribed into cDNA. The AtACTIN2 (NM_112764) was used as an internal reference gene. RT-PCR primers are presented in Table S9.
To validate the DEGs using RT-qPCR, 15 genes responding to salt stress were selected from the RNA-seq studies. Total RNA samples from SlD, SlG_C, and SlG_N were extracted as stated in the Plant Total RNA Purification Kit (Tiangen, Beijing, China). An amount of 1.5 ug RNA was converted into cDNA using the reverse transcription kit (TransGen Biotech, Beijing, China). RT-qPCR experiments were conducted using fluorescence quantitative PCR kit (TransGen Biotech, Beijing, China), and then analyzed via the Real-Time PCR machine (Takara, Japan). The cycle threshold (Ct) 2^−ΔΔCt method was used to calculate the relative expression levels of these genes. The SlACTIN (GenBank No. JX860282) was used as an internal reference gene. The primers for RT-qPCR are presented in Table S9.

The seed coat samples from different developmental stages (18 and 26 DAP) and other tissue samples, including roots, stems, leaves, and male flowers were collected from both parental lines. RNA was isolated using the plant total RNA purification kit (TIANGEN, China) according to the manufacturer’s instructions and then the first-strand cDNA was synthesized using a cDNA synthesis kit (Takara, Japan).
The gene-specific primers of the candidate genes and reference gene Actin (Kong et al., 2015 (link)) for quantitative real-time PCR (qRT-PCR) were designed based on the Cucurbit Genomic Database (http://cucurbitgenomics.org), using the software Primer Premier 5. The expression levels of the candidate genes were performed using a LightCycler480 RT-PCR system (Roche, Swiss) with a Real Master Mix (SYBR Green) kit (Toyobo, Japan). Amplification was carried out in a 20 µl reaction mixture containing 1 µl cDNA, 1 µl forward and reverse primers (10 µM), 10 µl 2 × SYBR Green real-time PCR mixes, with nuclease-free water added to a total reaction of 20 µl. Three biological and technical replicates were used for qRT-PCR. Average relative expression levels for each sample were calculated. The expression level was analyzed by the 2^−△△Ct method (Livak and Schmittgen, 2001 (link)), and the primer sequences used in this study are listed in Table S3.

Total RNA of 15 samples (Fig. S1) was extracted using a plant total RNA purification kit, following the manufacturer’s instructions (TIANGEN, China). The integrity and quantity of the RNA samples were analyzed by 1% agarose gel electrophoresis and with a NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). To analyze the transcriptome, qualified RNA samples were sent to Majorbio (Shanghai, China) to construct the cDNA library and then be sequenced on an Illumina HiSeq4000 (Illumina Inc., San Diego, CA, USA). Paired-end reads were generated and examined with the fastx_toolkit_0.0.14 software (http://hannonlab.cshl.edu/fastx_toolkit/) to assess the quality of those reads.