Comparative analysis of plastomes of Rubus cultivars and development of molecular markers for species delimitation.

Plastome analyses confirmed the taxonomic classification of cultivated Rubus and identified candidate InDel markers for distinguishing major cultivated groups, offering genetic resources for future studies of hybrid identification and breeding. The genus Rubus, belonging to the Rosaceae family, comprises numerous wild germplasm resources. Among its most economically valuable cultivars are red raspberry, black raspberry, blackberry, as well as raspberry × blackberry hybrids. In this study, we assembled and characterized 81 newly Rubus plastomes, including 22 wild species and 59 cultivars. All genomes exhibited the typical quadripartite structure, with sizes ranging from 155,385 to 157,012 bp and containing 130 annotated genes (113 unique functional genes), indicating a high degree of conservation in plastome architecture and gene order. A comparative analysis was further conducted using plastomes from 16 representative Rubus cultivars, representing all unique plastome sequences. The repeat sequences, codon usage bias, and hypervariable regions were systematically analyzed. Phylogenetic analysis revealed that modern Rubus cultivars primarily cluster within three subgenera: Rubus, Idaeobatus, and Melanobatus, with black raspberry phylogenetically placed within Melanobatus. Furthermore, four InDel markers were developed and validated for distinguishing three Rubus cultivar groups (black raspberry, red raspberry and hybrids). In summary, our research substantially expands available plastome resources for Rubus species and cultivars, thereby facilitating species delimitation and supporting future hybrid breeding.