Mitochondrial Genome Assembly and Comparative Analysis ofThree Closely Related Oaks

Quercus (oak) is an ecologically keystone and economically vital genus in Northern Hemisphere forests. Although genomic studies have advanced our understanding of its nuclear and chloroplast genomes, research on Quercus mitochondrial genomes (mitogenomes) remains limited due to their complex evolutionary dynamics, including extreme size variation, frequent rearrangements, and recurrent horizontal gene transfer(Christensen 2021). This study presents the assembly, annotation, and comparative analysis of mitogenomes from three closely related Asian oaks—Q. engleriana, Q. kongshanensis, and Q. tungmaiensis—using PacBio HiFi sequencing. Assemblies revealed distinct structural organizations: Q. engleriana and Q. kongshanensis mitogenomes each comprised one circular and one linear contig, while Q. tungmaiensis comprised one circular and two linear contigs. Total mitogenome lengths and protein-coding genes (PCGs) were determined as 445,351 bp (31 PCGs) for Q. engleriana, 538,610 bp (33 PCGs) for Q. kongshanensis, and 479,938 bp (38 PCGs) for Q. tungmaiensis, with similar GC content across species (45.6–45.9%). Comparative analyses identified variations in codon usage bias (RSCU), simple sequence repeats (SSRs; 154, 176, and 160, respectively), and predicted RNA editing sites (431–438 per species). Notably, RNA editing in rps12 was uniquely observed in Q. kongshanensis. Chloroplast-derived sequences (MTPTs) constituted 1.39%, 1.79%, and 2.24% of the mitogenomes, respectively. Phylogenetic reconstruction based on mitochondrial PCGs robustly resolved Q. kongshanensis and Q. tungmaiensis as sister species, with all three forming a distinct clade separate from other Quercus. This study provides comprehensive mitogenomic resources essential for elucidating Quercus evolutionary biology and supporting germplasm development.