*2.4. RNA Extraction and RT-qPCR Assays*

The total RNA was extracted using the RNAprep Pure Plant kit (Tiangen, Beijing, China), and its quality and concentration were determined using a NanoDrop 2000 spectrophotometer. cDNA synthesis was conducted by treating 1 g of total RNA using RNasefree DNase I and a reverse transcriptase, strictly following the guidelines given by the manufacturer (Thermo Fisher Scientific, Shanghai, China). We investigated the expression patterns of *GhSAMSs* under drought and salt stress at different time intervals, using previously released RNA-Seq data (https://cottonfgd.org/analyze/ accessed on 5 May 2020). According to the genes' expression patterns, we selected 14 genes, including 5 genes that were induced under salt and drought stress, 5 genes that were downregulated under the stress conditions, and 4 genes that showed similar expression under normal and stress conditions for the RT-qPCR analysis. Using the Premier Premier5 software, the primers of all the selected genes were designed for the RT-qPCR assays (Table S2). *GhActin* was chosen to serve as a standard reference gene. The SYBR Green Real-Time PCR Master Mix (Thermo Scientific, Rockford, IL, USA) was used to perform qPCR assays following the procedure described previously [34]. The reactions comprised the following reagents: cDNA template (5 μL), forward primer (0.5 μL), reverse primer (0.5 μL), SYBR green master mix (10 μL), and dd H2O (4 μL). The final mixture, whose concentration was at 10 mM, was centrifuged at 12,000 rpm for 1 min and placed into PCR thermal cycling conditions, as previously described [35]. The PCR procedure was performed according to the manufacturer's instructions. Pre-incubation, 1 cycle: 95 ◦C for 30 s; Amplification, 40 cycles: 95 ◦C for 10 s, 60 ◦C for 30 s; Melting curve, 1 cycle: 95 ◦C for 15 s, 60 ◦C for 60 s, 95 ◦C for 15 s; Cooling, one cycle: 40 ◦C for 30 s. The real-time analysis of each gene was performed with three independent biological replicates under the same conditions. The expression levels of the genes were analyzed using the 2−ΔΔCT method [36].
