Comparative chloroplast genomics of Medicago: codon usage bias, purifying selection, and phylogenomics

Medicago sativa subsp. falcata (yellow-flowered alfalfa) is an essential genetic resource for forage improvement, biofuel production, and sustainable agriculture. Its superior cold tolerance and drought resistance, compared with common alfalfa, make it suitable for cultivation in marginal and climate-stressed environments. Natural ploidy variation within this subspecies (diploid 2 n = 2 x = 16; tetraploid 2 n = 4 x = 32) provides a valuable system to investigate the effects of whole-genome duplication on organellar genome evolution, with direct implications for molecular breeding. Although accumulating chloroplast genome data have advanced evolutionary research across the genus Medicago , few comparative studies simultaneously integrate genus-wide datasets and focus on intraspecific cytotypes; specifically, the influence of polyploidization on plastomic features of M. sativa subsp. falcata remains poorly characterized. We sequenced and assembled the complete chloroplast genomes of diploid and tetraploid M. sativa subsp. falcata , and performed comparative genomic analyses across 30 Medicago species. The diploid chloroplast genome (125,934 bp) was slightly larger than the tetraploid (125,770 bp), with similar GC content (approximately 33.80%) and minor differences in gene counts. Codon usage bias (CUB) analyses revealed dominant A/U-ending preferred codons, shaped primarily by natural selection. Abundant simple sequence repeat (SSR) loci, mainly mononucleotides in non-coding regions, represent candidate resources for chloroplast‑derived marker development. Nonsynonymous (Ka) /synonymous (Ks) analysis revealed strong purifying selection (Ka/Ks < 0.5) across 77.1% of genes, particularly those involved in photosynthesis and energy metabolism. Phylogenetic reconstruction and collinearity analysis demonstrated high structural conservation among closely related species, with diploid and tetraploid M. sativa subsp. falcata clustering with other M. sativa accessions, confirming subspecies-level relationships. Polyploidization exerts limited influence on chloroplast genome size, gene content and structural organization, with strong functional constraints preserved across ploidy levels. The prevalent A/U codon preference and widespread purifying selection reflect conserved selective regimes acting on chloroplast coding sequences. This study delivers valuable chloroplast genomic resources and candidate SSR loci, which can facilitate germplasm evaluation, phylogenetic inference and marker-assisted breeding of Medicago forage cultivars.