With the increasing consumption of mulberry fruits in commercial markets, flavor profiles have emerged as critical determinants of consumer preference and market acceptance. This investigation utilized four Morus laevigata (Morus L.) accessions exhibiting pronounced variations in fruit pigmentation and flavor characteristics as experimental materials. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOF MS) was employed to identify key volatile aromatic compounds, while integrated untargeted metabolomics and transcriptomics approaches were applied to elucidate the underlying mechanisms of flavor biosynthesis. Analysis revealed that aldehydes, ketones, lactones, and heterocyclic compounds constitute the primary volatile organic compounds responsible for M. laevigata flavor complexity. The biosynthesis of these volatile aromatic compounds exhibits a direct correlation with lipid metabolite oxidation pathways. Concurrently, oxidative processes are modulated by M. laevigata flavonoid metabolites with antioxidant properties, which subsequently regulate both the compositional profile and quantitative distribution of volatile aromatic compounds. These findings offer novel insights into the metabolite–volatile compound interactions within mulberry systems, establishing a foundational framework for advancing fruit flavor research and cultivar development programs.

Mulberry (Morus spp.) includes ecologically important tree species that are highly valued for their exceptional economic and medicinal properties. Among its diverse species, Morus wittiorum and Morus laevigata are particularly valuable genetic resources because of their resistance to Sclerotinia, their relatively high content of specific flavonoids, and elongated fruit morphology. In this study, hybridization experiments were conducted using 10 mulberry accessions spanning three taxonomic sections (Alba, Wittiorum, and Laevigata). All six attempted intersectional crosses successfully yielded hybrid progeny. Using genomic in situ hybridization with blocking DNA, we detected distinct chromosomal signal patterns among the three sections, enabling precise identification of hybrid and wild-type chromosomal constitutions. Notably, this study provides the first documented evidence of 2n gamete formation in the genus Morus, where 2n eggs from M. wittiorum 'W-4' produced a pentaploid hybrid, 'Mp-7'. This discovery not only rewrites the chromosomal inheritance patterns of Morus but also unveils untapped polyploid breeding potential. These findings provide an efficient approach for identifying hybrids and offer novel polyploid breeding strategies, thereby promising to reshape global mulberry breeding and creating new opportunities for the genetic improvement of this agronomically important species.