Grapevine (Vitis vinifera) is economically important for fresh consumption, winemaking, and drying. The circadian systems of flowering and fruit development are crucial for viticulture and yield formation. However, the genetic basis of continuous flowering and bearing has been rarely elucidated. Here, we integrate pan-genomics, comparative genomics, single-cell transcriptomics, and bulk transcriptomics to investigate the continuous flowering and bearing trait (CFB) in the ‘Julian’ grape, which bears fruits at different development stages from flower to mature berries simultaneously. Pan-genomics and comparative genomics discovered 558 unique structural variations (SVs) and eight genes enriched in flowering pathways exclusively in Julian, based on the newly generated haplotype-resolved near telomere-to-telomere (T2T) genome of ‘Julian’ and 15 previously published genomes of grapevine, which bears fruits in the same developmental stage (i.e., seasonal flowering and bearing, SFB). Single-cell transcriptomes of flowering buds for CFB ‘Julian’ and SFB ‘Muscat Hamburg’ detected seven distinct cell types, which provide detailed cell-type-specific gene expression profiles for both cultivars, with differential gene expression (DEG) insights highlighting growth, metabolism, and hormonal regulation pathways in ‘Julian’. Integrating SVs and DEG data, we pinpoint 37 candidate genes potentially associated with CFB, including Auxin/IAA, Cytochrome P450, Vicilin-like antimicrobial peptides (AMP) and respiratory burst oxidase homolog protein B (RBOH) related genes. This study provides insights into the genetic basis of CFB in grapevine, facilitating grapevine breeding with continuous flowering and bearing.

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