Comparative chloroplast genomics of four Actinidia species: structure, variation, and phylogeny

Abstract Background Actinidia Lindl. represents the most economically and nutritionally valuable genus within Actinidiaceae. However, species identification and intraspecific discrimination in this genus remain to be fully clarified, in particular, the genetic relationships and fine-scale variations within section Leiocarpae are still poorly understood. To address this, we assembled and annotated the chloroplast genomes of eight accessions, representing four species in this section: A. arguta , A. melanandra , A. kolomikta , and A. polygama . Result All eight chloroplast genomes exhibited remarkable conservation in structural organization and gene composition, with sizes varying between 156,661 and 157,614 bp and a uniform GC content of 37.20%. Long repeat sequences were mainly of the forward, palindromic, and reverse, while complementary repeats were absent. A large number of simple sequence repeats (SSRs) were also identified, among which mononucleotide repeats were the most abundant. Comparative analysis revealed seven variable regions ( rps12-psbB , trnN-ndhF , rbcL-accD , petA-psbJ , ndhF , ycf1 , and ycf2 ). PCR validation confirmed that the rbcL-accD variable region could distinguish the four studied species, indicating its strong potential as a novel marker for Actinidia . Codon usage bias analysis revealed a preference for codons ending in A/T in these genomes. Phylogenetic analysis based on the cp genome revealed that all accessions were resolved into two major clades, corresponding to section Leiocarpae and non- Leiocarpae taxa, and these two clades were further validated by SNP-based PCA, haplotype network, and population structure analyses. Strikingly, different accessions within the same species, particularly the newly sequenced A. arguta accessions, were placed on distinct lineages, revealing more complex relationships within Leiocarpae . Conclusion Our assembled cp genomes enrich Actinidia genomic resources and provide new insights into the phylogenetic relationships of the genus. We also characterized variable regions within section Leiocarpae and experimentally validated the potential of the rbcL–accD region as a molecular marker for distinguishing selected species. The observed intraspecific divergence, particularly among A. arguta accessions, highlights the complex evolutionary history of section Leiocarpae and may reflect shared paternal ancestry and/or historical introgression among closely related species. Overall, these findings advance our understanding of chloroplast genome evolution and provide useful resources for species identification, germplasm characterization, and phylogenetic studies in Actinidia .