Phased T2T genome of a tetraploid grapevine reveals segmental allopolyploid origin and allele-specific alternative splicing during fruit development

The tetraploid table grape Kyoho, a cultivar of major global economic importance, has long presented a challenge in understanding its genetic architecture and the regulatory landscapes between its different ancestral genomes. Here, we assemble a haplotype-resolved, near-complete reference genome (480.32-489.47 Mb, contig N50 17.5-23.3 Mb, with a total of only 11 gaps) to investigate regulatory dynamics through integrative multiomic analyses during fruit development. Comparative evolutionary genomics classifies Kyoho as a segmental allotetraploid with a complex mosaic genome, composed of ∼71% V. vinifera (Vv) and ∼29% V. labrusca (Vl) ancestry. A total of 67.3% of genes are shared among the four haplotypic genomes with 9.8% haplotype specific genes. Transcriptomic analysis revealed that among 17,750 pairs of ancestral alleles exhibiting directional bias, only 4,086 (23.4%) maintained a consistent ancestral dominance direction across all tissues. Similarly, comparisons of DNA methylation profiles showed no significant differences between genome ancestries Vl (0.009∼0.676): Vv (0.010∼0.670). However, alternative splicing (AS) analyses demonstrated that allele-specific splicing changes were correlated with ancestral origin across tissues (Vl: 1,529 vs. Vv: 2,052). Integrative omics identified 2,307 differentially expressed ancestral alleles that showed no significant genomic variants or methylation bias but exhibited AS bias. These genes include key regulators of fruit ripening, including ZEP, PIN, DOG1and MYB. Overall, this work delivers a landmark genomic resource and characterizes its regulatory architecture, facilitating functional genomic studies and guiding precision polyploid breeding in grapes.