*5.9. RNA Isolation and Transcriptional Profiling of GhPP2C under Various Stresses*

Total RNA was isolated from the treated frozen leaves with Trizol (Invitrogen) following the manufacturer's instructions. RNA was reverse-transcribed into cDNA using the Primer Script RT reagent kit (TAKARA, Dalian China) according to their instructions. Specific primers were designed using Becan Designer 7.9 and are presented in Supplementary Table S7. In order to check the specificity of the primers, we used the BLAST tool against the *Gossypium hirsutum* genome for confirmation. RT-PCR was performed according to the guidelines of previous studies [54]. Relative fold expression was calculated with the comparative Ct-method. The expression patterns of all *GhPP2C* genes were analyzed based on a previous study [55,56]. The cotton histone3 (AF024716) gene was used as the reference gene for qRT- PCR. In brief, the real-time PCR amplification reactions were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, California, CA, USA) using SYBR Green (Vazyme, Nanjing, China) with three replicates. The amplification parameters were denaturation at 95 ◦C for 10 min, 40 cycles of denaturation at 95 ◦C for 15 s, annealing at 60 ◦C for 15 s, and extension at 72 ◦C for 15 s.

High-throughput RNA-sequencing data [24] was utilized for various vegetative (root, stem, and leaf), floral (stamen and patel), and fiber tissues (3, 6, 9, 12, and 15 days post anthesis), respectively. Furthermore, gene expression levels were quantified by FPKM (fragments per kilobase of transcript per million fragments mapped) values, and heat maps were generated using an online omicshares tool (http://www.omicshare.com/) and TBtools software [52].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/20/6/1395/ s1.

**Author Contributions:** Conceptualization and methodology, writing—original draft preparation H.S.; review and editing, N.K.; software, J.W.; validation, C.W., Z.W., and Z.H.; supervision, X.W.

**Funding:** This study was financially supported by National Key Research and Development Program for Crop Breeding (2017YFD0102000) and Natural Science Foundation in Jiangsu Province (BK20160712).

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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