Comparative Chloroplast Genomes Analysis Provided Adaptive Evolution Insights in Medicago ruthenica

A perennial leguminous forage, Medicago ruthenica has outstanding tolerance to abiotic stresses. The genome of Medicago ruthenica is large and has a complex genetic background, making it challenging to accurately determine genetic information. However, the chloroplast genome is widely used for researching issues related to evolution, genetic diversity, and other studies. To better understand its chloroplast characteristics and adaptive evolution, chloroplast genomes of 61 Medicago ruthenica were assembled (including 16 cultivated Medicago ruthenica germplasm and 45 wild Medicago ruthenica germplasm). These were used to construct the pan-chloroplast genome of Medicago ruthenica, and the chloroplast genomes of cultivated and wild Medicago ruthenica were compared and analyzed. Phylogenetic and haplotype analyses revealed two main clades of 61 Medicago ruthenica germplasm chloroplast genomes, distributed in eastern and western regions. Meanwhile, based on chloroplast variation information, 61 Medicago ruthenica germplasm can be divided into three genetic groups. Unlike the phylogenetic tree constructed from the chloroplast genome, a new intermediate group has been identified, mainly consisting of samples from the eastern region of Inner Mongolia, Shanxi Province, and Hebei Province. Transcriptomic analysis showed that 29 genes were upregulated and three genes were downregulated. The analysis of these genes mainly focuses on enhancing plant resilience and adapting adversity by stabilizing the photosystem structure and promoting protein synthesis. Additionally, in the analysis of adaptive evolution, the accD, clpP and ycf1 genes showed higher average Ka/Ks ratios and exhibited significant nucleotide diversity, indicating that these genes are strongly positively selected. The editing efficiency of the ycf1 and clpP genes significantly increases under abiotic stress, which may positively contribute to plant adaptation to the environment. In conclusion, the construction and comparative analysis of the complete chloroplast genomes of 61 Medicago ruthenica germplasm from different regions not only revealed new insights into the genetic variation and phylogenetic relationships of Medicago ruthenica germplasm, but also highlighted the importance of chloroplast transcriptome analysis in elucidating the model of chloroplast responses to abiotic stress. These provide valuable information for further research on the adaptive evolution of Medicago ruthenica.