Comparative Chloroplast Genomics Reveals the Maternal Origin and Evolutionary Relationships of Commercial Pluot Cultivars Within Prunus

Background: The phylogenetic placement and chloroplast-inferred maternal relationships of commercial pluot cultivars remain unclear, largely because plastome-level evidence is limited for assessing their affinities with Prunus salicina and Prunus ussuriensis. Although chloroplast genome structure has been well characterized in angiosperms and in several Prunus species, complete plastome resources and comparative genomic evidence for commercial pluot cultivars remain scarce. Methods: Here, we assembled the complete chloroplast genomes of six commercial pluot cultivars and performed comparative genomic, phylogenomic, and divergence time analyses using representative Prunus species. Results: All genomes exhibited the typical circular quadripartite structure and ranged from 157,865 to 158,138 bp in length. Genome organization, GC content, and gene content were highly conserved, whereas the IR regions showed an elevated GC content of approximately 42.6%, owing to rRNA gene enrichment. IR boundary comparison revealed contraction at the IRb/SSC boundary in P. ussuriensis, while pluot cultivars were structurally more similar to P. salicina. In total, 370 SSR loci and four hypervariable regions, namely rpoB–trnC-GCA, petN–psbM, trnV-UAC–trnM-CAU, and trnP-UGG–psaJ, were identified as candidate molecular markers for Prunus germplasm identification and genetic analysis. Phylogenomic analysis resolved four major clades within Prunus and showed that ‘Flavor King’, ‘Flavor Supreme’, and ‘Flavor Queen’ grouped with P. ussuriensis, whereas ‘Flavorosa’, ‘Dinosaur Egg’, and ‘Flavorich’ grouped with P. salicina. Conclusion: Overall, this study provides the first comparative plastome analysis of six commercial pluot cultivars and offers chloroplast-level evidence for their maternal affinities within Prunus, together with useful marker resources for cultivar identification and germplasm evaluation.