Complete assembly of Onobrychis viciifolia mitochondrial genome reveals evolutionary dynamics.

This study presents the mitogenome characteristics analysis of Onobrychis viciifolia, including anatomization of repeat sequences, RNA editing site prediction, substitution rate estimation, and phylogenetic and collinearity analyses. Onobrychis viciifolia is a perennial forage legume with high crude protein content. Although its chloroplast genome has been characterized, the mitochondrial genome (mitogenome) had not previously been annotated. In this study, we assembled the first complete mitogenome of O. viciifolia using a hybrid approach, integrating Illumina NovaSeq short-read and Nanopore PromethION long-read data. The single master circular mitogenome spans 351,303 bp (GC content: 45.17%) and encodes 31 protein-coding genes (PCGs), 20 transfer RNAs, and 4 ribosomal RNAs. Through structural analysis, we identified 76 simple sequence repeats, 144 dispersed repeats, and 6 tandem repeats. Codon usage analysis of 9533 codons revealed a pronounced bias for TTA-encoded leucine (relative synonymous codon usage = 1.53). The analysis of synonymous (Ks) and nonsynonymous (Ka) substitution rates (Ka/Ks) of 21 core genes from 20 legume species revealed that the majority of genes were subject to purifying selection, whereas potential positive selection signals were detected in the ccmB and nad9 genes. We predicted 459 RNA editing sites, with nad4 and ccmB exhibiting higher editing frequencies of 45 and 34 events, respectively. Comparative mitogenomic analysis detected 37 chloroplast-derived sequences (total length: 11,539 bp; 3.28% of the mitogenome), including four transfer RNA genes (trnD-GUC, trnH-GUG, trnM-CAU, and trnF-GAA). Phylogenetic analysis indicated that O. viciifolia and Astragalus complanatus are most closely related, with the node supported by a bootstrap value of 100%, which was corroborated by the phylogenetic tree constructed from 23 plant species. Collinearity analysis further revealed the highly rearranged nature of its mitochondrial genome structure. The assembled and annotated mitogenome provides a valuable resource for investigating cytoplasmic inheritance mechanisms.