Winterberry (Ilex verticillata) is a deciduous shrub within the Aquifoliaceae family that holds significant ornamental and medicinal value. However, the lack of systematic research on the genetic background and phylogenetic relationships among its cultivars has hindered germplasm conservation and breeding efforts. This study marks the first application of genotyping-by-sequencing (GBS) technology to analyze winterberry germplasm resources. Sequencing was performed on 79 samples from eight representative cultivars, and 3,411,968 high-quality single-nucleotide polymorphism (SNP) markers were developed using a de novo assembly strategy. Population structure analysis based on STRUCTURE indicated K = 8 as the statistically optimal number of genetic components according to the delta K statistic. However, when STRUCTURE results were interpreted together with principal component analysis (PCA) and phylogenetic reconstruction, the winterberry cultivars were consistently summarized into five major genetic clusters. Ilex verticillata ‘Winter Gold’ and I. verticillata ‘Winter Red’ shared highly consistent genetic backgrounds, indicating extremely close kinship; I. verticillata ‘Citronella’ and I. verticillata ‘Oosterwijk’ clustered closely together; I. verticillata ‘Red Sprite’ (Rizhao) and I. verticillata ‘Red Sprite’ (Dezhou), despite differing geographical origins, clustered together, demonstrating good genetic stability; and I. verticillata ‘Golden Verboom’ and I. verticillata ‘Little Goblin Red’ each formed independent genetic branches, possessing unique genetic backgrounds. This study concludes that GBS effectively reveals the complex genetic structure among winterberry cultivars. The findings not only provide accurate molecular evidence for cultivar identification and intellectual property protection but also lay a solid foundation for future hybrid breeding, including parent selection, identification of superior genes, and advancement of marker-assisted breeding.

Ilex rotunda Thunb. is a prestigious ornamental tree renowned for its vibrant red fruits, yet the molecular mechanisms governing its fruit color variation remain poorly understood. The discovery of a rare yellow-fruited natural bud sport cultivar, ‘Peace Time’, provides an ideal model to investigate these processes compared to the wild-type red fruit. In this study, we integrated physiological evaluations, untargeted metabolomics, and de novo transcriptomics across multiple fruit developmental stages to elucidate the basis of this color transition. Our results demonstrated that the yellow phenotype is characterized by high lightness and yellowness values, driven by the profound suppression of anthocyanin biosynthesis. Biochemical and transcriptomic profiling revealed that DFR (dihydroflavonol 4-reductase), a critical “gatekeeper” gene, experiences severe transcriptional silencing in the yellow-fruited cultivar. This enzymatic bottleneck triggers a “passive substrate overflow,” redirecting shared precursors toward the parallel flavonol branch, resulting in the substantial accumulation of specific flavonols, including rutin and isoquercitrin. Furthermore, correlation network analysis highlighted a putative dual regulatory module associated with this metabolic reprogramming: the down-regulation of the putative activator bHLH30 coupled with the robust up-regulation of the putative repressor bHLH51, together likely contributing to the silencing of DFR transcription. These findings provide a comprehensive “dual-module” and “passive overflow” framework for fruit coloration in I. rotunda, highlighting a remarkable metabolic plasticity that reshapes this cultivar’s phytochemical profile and offers vital insights for future ornamental breeding.