Single-nucleus transcriptome atlas of Brassica developing seeds provides cellular and molecular insights into embryo abortion in interspecific hybrids

Embryo rescue has been used to recover abortive seeds in distant hybridization; however, its efficiency remains low and rescue frequently fails because the genetic basis of embryo abortion is poorly understood. To address this, time-series single-nucleus RNA sequencing (snRNA-seq) was performed on developing seeds from interspecific crosses between Brassica campestris (syn. Brassica rapa) ssp. chinensis and Brassica oleracea, and compared them to seeds from B. campestris intraspecific crosses. Our analysis of single-nucleus transcriptomic profiles revealed that during normal seed development, embryo and endosperm cells undergo robust proliferation, whereas in interspecific hybrids, both cell types are drastically reduced, suggesting an early and ongoing developmental failure. We identified a previously uncharacterized cell type designated seed coat and suspensor (SC-SUS), characterized by the unique expression of CYP78A6, a cytochrome P450 family gene. Notably, CYP78A6 is deactivated after the globular stage in normal seeds but remains active in hybrid seeds. Analysis of hormone regulatory genes revealed substantial disruption of hormonal balance in hybrids. Additionally, mapping of global cell-cell communication networks mediated by ligand-receptor pairs, revealed extensive dysregulation in interspecific seeds. Cross-species validation in Arabidopsis demonstrated that both maternal CYP78A6 mutation and exogenous hormone application restored hybrid seed viability, with the mutation exhibiting a stronger effect, and their combination provided a modest additional advantage. This study establishes a high-resolution cellular and genetic framework for understanding interspecific embryo abortion, reveals that reproductive isolation can be explained by genetically programmed cell-type-specific programs, and identifies potential genetic targets for improving embryo rescue in crop breeding.