Genomic divergence, introgression and KASP fingerprinting panel development in five cultivar groups of sweet osmanthus

Sweet osmanthus is a well-known ornamental tree endemic to China. Its prolonged history of domestication, accompanied by inter-specific gene flow and frequent selection of bud mutations, has given rise to over 200 cultivars categorized into five cultivar groups. However, there is still a lack of information on population genetic composition and high-throughput genotyping tools for capturing the genomic divergence of accessions across the five cultivar groups. Here we performed whole genome resequencing on both cultivated sweet osmanthus and wild Osmanthus accessions to explore their phylogenetic relationship and wild-to-cultivar introgression. Phylogenetic analysis revealed that O. fragrans cultivars formed a monophyletic clade and were further delineated into seven distinct genetic groups. This genetic structure was discordant with the classification of traditional cultivar groups based on floral traits. Luteus and Albus are more original, and Caiyegui exhibited a relatively narrow genetic background (π = 2.8 × 10⁻³). Gene flow estimates revealed introgression (18,734 significant trios in D-statistics) from autumn-flowering species in O. sect. Osmanthus to four sweet osmanthus cultivars. Through rigorous filtering, 571 SNPs were selected from 9.36 million SNPs for kompetitive allele specific PCR (KASP) marker design and large-scale genotyping. By establishing a core marker selection pipeline, we retained a core set of 143 markers with 69.9% located in genic regions. They were verified to be efficient in identification and fingerprinting of 163 cultivars, including the newly-bred and inter-specific hybrids. To our knowledge, this study provides the first genomic evidence suggesting that interspecific introgression may have played a role in the development of a specific subset of O. fragrans cultivars (the G1 group), and also offers high-throughput genotyping tool that will greatly facilitate molecular Distinctness, Uniformity, and Stability (DUS) testing and molecular breeding in sweet osmanthus.