*5.1. Data Resources for Sequence Retrieval*

For identification of *PP2C* genes in *Gossypium* and other species, we utilized the Plaza 4.0 database (https://bioinformatics.psb.ugent.be/plaza/) with the help of InterPro PP2C domain "IPR001932". The cotton genome sequences were downloaded from (https://www.cottongen.org/), and *A. thaliana*

sequences were retrieved from TAIR (http://www.arabidopsis.org/). The domains of obtained GhPP2C proteins were further verified using the NCBI-Conserved Domain database (https://www. ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) search program and SMART databases (http://smart. embl-heidelberg.de/) [46]. Those proteins which lack PP2C domains were removed from further analysis. In addition, protein sequences that were found with obvious errors in their gene length or having less than 100 lengths were eliminated.

#### *5.2. Multiple Sequence Alignment and Phylogenetic Analysis*

The amino acid sequences of the GhPP2C proteins were used for further investigation, and multiple sequence alignment was performed by MUSCLE [47] using MEGA 7 software with the default options [48]. The phylogenetic trees were constructed using the maximum likelihood (ML) method. In order to determine the reliability of the resulting tree, bootstrap values of 1000 replications were performed with the Jones, Taylor, and Thornton amino acid substitution model (JTT model), while keeping the other parameters as a default.

#### *5.3. Calculation of the Ka/Ks for Duplicated Genes*

The *Ka/Ks* ratios were calculated for duplicated (segmental and dispersed) gene pairs using MEGA 7.0 [48].

#### *5.4. Conserved Motifs, Exon-Intron Structure Analysis, and Physicochemical Parameters of PP2C Proteins*

Conserved motif scanning of GhPP2C proteins was carried out through local MEME Suite Version 5.0.3. For this purpose, parameter settings were calibrated as follows: Maximum number of motifs 10, with a minimum width of 100 and a maximum of 150. The other parameters were set as default [49]. For the exon-intron structure, we used the Gene Structure Display Server (GSDS 2.0) (http://gsds.cbi. pku.edu.cn) [50]. The physicochemical properties of the proteins, including molecular weight (MW), isoelectronic points (pI), aliphatic index, and GRAVY values for each gene, were calculated using the ExPASY PROTPARAM tools (http://web.expasy.org/protparam/). The subcellular localization was predicted using the WOLF PSORT (https://wolfpsort.hgc.jp/) website.

## *5.5. Cis-Elements Predictions of GhPP2C*

Every GhPP2C promoter sequence (selected as 2000 upstream bp) was imported in Generic File Format (GFF) file from the cotton genome. Then, the PlantCARE database (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/) [51] was utilized to identify the cis-regulatory elements for promoters of each gene.

#### *5.6. Chromosomal Location and Synteny Correlation Analysis*

The chromosomal location of *PP2C* genes was illustrated from top to bottom concerning their position in the genome annotation by using TBtools software [52]. For synteny gene analysis, the relationships were verified between the homologs of A. *thaliana* and *Gossypium hirsutum*. Circos (using TBtools software) program was applied to exhibit the syntenic relationships among the chromosomes of *G. hirsutum* and *A. thaliana* [52].

#### *5.7. Pearson Correlation Analyses (PCC)*

Pearson correlation analysis was performed with the help of Excel 2013 in order to evaluate the PCC values that were used for qRT-PCR according to a previously reported study [53]. As well, the PCC of fragments per kilobase of transcript per million fragments mapped (FPKM) values was implemented using RStudio (R program) at 0.05 (*p*-value) significance level.
