Comparative chloroplast genome analysis of four Trigonella species: structural rearrangements, gene loss, and phylogenetic relationships.

Fabaceae is one of the most structurally dynamic plant families for chloroplast genome evolution, particularly within the inverted repeat-lacking clade (IRLC), where the loss of one inverted repeat predisposes plastomes to accelerated rearrangement and gene loss. Here, we present the complete chloroplast genomes of four Trigonella species, T. foenum-graecum, T. corniculata, T. caerulea, and T. gladiata, and perform a comprehensive comparative analysis of their plastome evolution and phylogenetic relationships. All four genomes range from 124,823 to 125,437 bp, each encoding 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. Three genes, infA, rpl22, and rps16, are retained as pseudogenes in one or more species, capturing distinct stages of ongoing plastid-to-nucleus gene transfer. Whole-genome alignment revealed a complex structural rearrangement in the T. foenum-graecum plastome, involving the inversion and translocation of four locally collinear blocks across a ~35.5 kb region, a pattern absent from the three collinear species. Most protein-coding genes evolve under strong purifying selection; five genes (clpP, ycf2, cemA, ycf1, and rpl20) had Ka/Ks > 1, with only clpP reaching statistical significance. Nucleotide diversity analysis identified clpP as the most variable coding sequence, and the ycf2-trnI-CAU spacer, trnR-UCU-atpA, and rps15-ycf1 as the most variable intergenic regions. SSR analysis identified 101 polymorphic loci with associated primer pairs, providing resources for population-level studies. Phylogenetic analysis confirmed that Trigonella is not a natural group, with T. corniculata resolving as sister to Melilotus albus and M. officinalis with maximum bootstrap support, providing strong evidence for a formal taxonomic revision of the genus.