Dissecting the Evolutionary Forces Behind Codon Usage Bias in Brassica: A Chloroplast Genome Perspective from Non-Heading Chinese Cabbage

Chloroplast-genome variation below the species level remains insufficiently characterized in Brassica, because most previous studies have relied on only one or a few representative plastomes. To determine whether dense within-subspecies sampling could reveal localized variation obscured by broader comparisons, we sequenced and assembled complete chloroplast genomes from 10 morphologically diverse varieties of Non-heading Chinese cabbage (NHCC; Brassica campestris [syn. Brassica rapa] ssp. chinensis) and placed their codon-usage patterns in the context of 15 Brassica plastomes. The NHCC plastomes were highly conserved in genome size (153,480–153,533 bp), GC content (36.36–36.39%), gene content, gene order, and quadripartite structure. Nevertheless, locus-level comparisons showed that the limited sequence variation was concentrated in a small number of regions. Six protein-coding loci and four intergenic spacers exhibited relatively elevated nucleotide diversity, and explicit counts of variable sites and internal indel blocks further characterized the sequence differences underlying these values. These loci represent candidate regions for population-level validation and potential molecular-marker development rather than confirmed variety-specific markers. In the broader comparison, mean ENC varied only from 45.15 to 45.49 among the 15 plastomes, indicating a highly conserved codon-usage background characterized by a general preference for A/U-ending synonymous codons. Eight codons—ACU, AGA, CAA, CUA, GCU, GGU, GUA, and UGU—were consistently recovered as putative optimal codons, although statistical support for ACU and GGU was restricted to a subset of accessions. PR2 analysis revealed imbalances within complementary nucleotide pairs rather than an overall preference for G/C-ending codons. Thus, the principal contribution of this study is the fine-scale localization of plastome variation through dense sampling within NHCC, providing candidate regions for subsequent diversity assessment and marker validation in independently sampled individuals.