3.3. Chromosomal Localization, Gene Duplication, and Phylogenetic Analysis

The chromosomal locations of cotton CNGC genes were determined according to the genome annotation data; the positions and relative distances on the chromosomes were visualized using TBtools software [98]. Synteny, collinearity, and gene duplication were analyzed using MCScanX (http://chibba.pgml.uga.edu/mcscan2/ (accessed on 6 June 2021)) [41]. Gene duplication was determined according to two criteria: (a) the length of the shorter aligned sequence covered > 70% of the longer sequence, and (b) the two aligned sequences shared > 70% amino acid sequence similarity [99]. Two genes separated by less than five intermediate genes in the 100 kb chromosomal fragment are considered to have undergone tandem duplication [100]. To detect the mode of selection forces acting on the protein, the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) was calculated using TBtools with the Nei–Gojobori model [101]; generally, a Ka/Ks ratio > 1 indicates positive selection, the ratio < 1 implying negative or purifying selection, while the ratio = 1 indicates neutral evolution [43]. The density plots of Ks were analyzed and visualized using the lattice package in RStudio [102]. The estimated divergence time (T) of each duplicated gene pair was calculated as T = Ks/2r, in accordance with the neutral substitution rate of cotton (r = 2.6 × 10⁻⁹) [31]. The synteny relationship of orthologous CNGC genes in different species was constructed using the Dual Synteny Plotter (https://github.com/CJ-Chen/TBtools (accessed on 8 June 2021)) [103], and the results were visualized and optimized via RCircos [104]. The phylogenetic analysis was performed with the full-length amino acid sequences of CNGCs using MEGA X software (http://www.megasoftware.net/ (accessed on 4 July 2020)), wherein multiple sequence alignment was conducted using the ClustalW and the phylogenetic tree was generated using the neighbor-joining (NJ) method with 1000 bootstraps.