S-locus is the key locus in Brassica that controls the recognition and initiation of the self-incompatibility response. To clarify the codon usage characteristics of the S domain of S-locus genes SRK and SLG as well as of SP11/SCR coding sequence in Brassica oleracea, the codon usage bias of 41, 36, and 11 alleles of SRK, SLG, and SP11/SCR were analyzed by Codon W, SPSS, Python, and EMBOSS online tools, respectively. Neutrality, ENC-GC3, and Parity Rule 2 (PR2) plotting together with multivariate statistical analysis were also used for the exploration of possible factors that might affect the formation of the codon usage bias of these genes. The cluster analysis performed using various methods showed that S domains of SRK, SLG, and SP11/SCR genes of Brassica oleracea were rich in AT base pairs, the codon preference ended with A/T base and had a lower codon usage bias, which were affected by the natural selection (a main factor) and the mutational pressure (a secondary factor) as well as dinucleotide abundance. Based on the RSCU values, 4 over-expressed codons of SRK and SLG, and 11 over-expressed codons of SP11/SCR were identified. Cluster analysis based on RSCU could accurately reflect the relationship among SRK, SLG, and SP11/SCR alleles in Brassica oleracea, which was consistent and reliable with CDS sequence clustering. According to codon usage bias and cluster relationship, the S domain of SRK and the SP11/SCR coding sequence might be coevolutionary in the codon usage bias. This study enhances our understanding of the mechanism of distribution of codons and the evolution of SRK, SLG, and SP11/SCR genes in Brasscia oleracea.
