Haplotype-resolved telomere-to-telomere reference genome of sweet cherry ‘Tieton’ v3.0 characterized the large fragment deletion associated with yellow-skinned variety

Sweet cherry (Prunus avium) represents a significant stone fruit crop in temperate regions worldwide. While molecular breeding has progressed substantially following the initial sweet cherry genome release, existing genome assemblies contain unresolved gaps and comprise consensus chimeric sequences that fail to differentiate haplotype alleles, significantly constraining research on important agronomic trait inheritance. This study presents a phased-resolved telomere-to-telomere reference genome of sweet cherry ‘Tieton’. The assembly anchors 653.03 Mb of sequence onto 16 pseudochromosomes representing two haplotypes, with 67,012 coding genes identified (33,777 in hapA and 33,235 in hapB). The genome demonstrates superior quality metrics, including a consensus accuracy exceeding QV44, contig N50 above 17.94 Mb, Benchmarking Universal Single-Copy Orthologs completeness of 98.7%, and a long terminal repeat (LTR) assembly index exceeding 20. This genome provides phased and annotated chromosome pairs, offering a comprehensive view of sweet cherry’s diploid genome organization. Utilizing this reference genome, we identified a large fragment deletion associated with yellow-skinned fruit in sweet cherry ‘13-33’. This resource will significantly enhance breeding efforts and genetic research in sweet cherries.