Integrative chloroplast omics in citrus: computational insights into genomic structural adaptation and phylogenetic relationships from 35 species

The chloroplast genome serves as a valuable tool for plant phylogenetic studies due to its conserved structure and slow evolutionary rate. Despite the economic importance of the genus <i>Citrus</i>, comprehensive comparative analyses of chloroplast genomes across multiple species remain limited. As one of the ancestors of <i>Citrus</i>, <i>Citrus medica L</i>. serves as a key representative in studies of the origin and evolution of the genus. In this study, we conducted a comprehensive comparative analysis of chloroplast genomes from 35 Citrus species and their close relatives, with <i>C. medica</i> as a focal species, to investigate structural variation, codon usage patterns, and phylogenetic relationships. All genomes exhibited a typical quadripartite structure, ranging from 159 to 161 kb with GC contents of 38.41-38.49%. While genome synteny was highly conserved, expansions and contractions at IR boundaries provided species-specific variation. SSR analysis revealed abundant mononucleotide repeats with a strong AT bias, predominantly distributed in non-coding regions. Codon usage analysis indicated a preference for A/U-ending codons, and ENC-GC3s, neutrality plot, and PR2 analyses suggested that natural selection was the main force shaping codon usage bias. Most protein-coding genes were under strong purifying selection, whereas <i>matK</i> and <i>rps16</i> exhibited elevated Ka/Ks ratios, suggesting relaxed selective constraints or potential signals of positive selection. Phylogenomic analysis strongly supported the monophyly of <i>Citrus</i> and resolved intrageneric relationships, grouping species into distinct ancestral and cultivated clades. Overall, this study provides essential chloroplast genomic resources for molecular breeding, species identification, and understanding <i>Citrus</i> adaptation.