A research team has produced the first gap-free, telomere-to-telomere (T2T) reference genome for broccoli and used it to uncover a major genetic switch behind purple bud coloration. The assembly closes longstanding gaps across all nine chromosomes, giving researchers a clearer view of regions that were previously difficult to resolve. Guided by this complete map, the team identified BoF3’H as a key regulator of anthocyanin production and showed that disrupting the gene sharply reduced purple pigments and turned buds green. The work links genome completeness directly to trait discovery, offering a practical foundation for breeding broccoli with tailored color, nutritional quality, stress resilience and market appeal for consumers and growers worldwide.
Broccoli is valued for vitamins, fiber, sulforaphane and anthocyanins, pigments that contribute to both nutritional properties and colors ranging from green to deep purple. Yet the crop’s highly repetitive genome has remained difficult to assemble, leaving gaps that can obscure genes, regulatory regions and structural variation. Earlier broccoli references improved chromosome coverage but still contained missing or unanchored sequences, limiting precise mapping of commercially important traits. At the same time, the genetic control of bud pigmentation has remained incomplete, even though bud color strongly affects commercial value and anthocyanins may support plant stress responses. Given these challenges, deeper investigation is needed into complete broccoli genome architecture and the genes controlling anthocyanin accumulation.
Researchers from the Institute of Horticulture, Shanghai Academy of Agricultural Sciences, and the College of Horticulture, Shenyang Agricultural University, reported (DOI: 10.1093/hr/uhag110) the study in Horticulture Research on April 21, 2026. They combined Oxford Nanopore Technologies ultralong sequencing, Pacific Biosciences high-fidelity sequencing and high-throughput chromosome conformation capture (Hi-C) to assemble the SN60 genome of broccoli ( Brassica oleracea var. italica ). This integrated strategy produced the crop’s first telomere-to-telomere (T2T) reference and enabled fine mapping and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) validation of BoF3’H , connecting a complete chromosome map with the molecular basis of purple bud formation.
The 633.61-megabase assembly spans nine gap-free chromosomes, includes all 18 telomeres and nine centromeres, and reaches a contig N50 of 60.36 megabases. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis recovered 99.4% of conserved plant genes, while annotation identified 58,044 protein-coding genes. Compared with earlier references, the assembly increased continuity roughly fourfold over BOP04-28-6 and filled all 97 gaps found in HDEM. It also enabled the researchers to catalogue more than 2.2 million single-nucleotide polymorphisms and insertions/deletions, together with 26,977 structural variants, between SN60 and BOP04-28-6. Using the new reference, the team crossed purple-budded BT126 with green-budded SN60 and applied bulked-segregant analysis (BSA) to locate a pigmentation region on chromosome C09. Fine mapping narrowed the locus to 73 kilobases, where BoF3’H , encoding flavonoid 3′-hydroxylase, emerged as the sole candidate. A natural 43-base-pair deletion in the green parent introduced a premature stop codon and lowered gene expression. CRISPR–Cas9 knockout lines then provided functional confirmation: homozygous bof3’h plants developed green buds, and ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) showed an 81.4% decrease in total anthocyanins, including 64.1% less cyanidin and 97.2% less delphinidin. Ribonucleic acid (RNA) sequencing further revealed coordinated suppression of the anthocyanin pathway.
The authors said the study shows why completing a crop genome matters beyond improving assembly statistics. A gap-free reference made it possible to move from an unresolved color trait to a precisely mapped gene and then test that gene directly in broccoli. They said BoF3’H now offers a clear biological entry point for understanding how purple pigments are produced, while the full genome provides a dependable framework for exploring other traits hidden in repetitive or structurally complex regions. Together, these resources connect chromosome-scale discovery with practical, more precise crop improvement.
The new reference could accelerate marker-assisted selection, genome editing and comparative genomics across broccoli and related Brassica crops . Breeders may use BoF3’H and linked markers to develop varieties with higher anthocyanin content, stable green coloration or distinct market-oriented appearances, depending on production goals. The genome also supports more accurate detection of structural variants associated with stress tolerance, yield and nutritional quality. Because complete telomere and centromere sequences are now available, researchers can examine chromosome regions often missed in draft assemblies and design editing targets with greater confidence. More broadly, the study provides a reusable genomic platform for connecting vegetable diversity with molecular mechanisms, precision breeding and future stress-resilience strategies across diverse production environments.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhag110
Funding information
This study was supported by the Shanghai Agricultural Science and Technology Innovation Program (grant no. K2023008) and the Excellent Team Project (Nongkezhuo 2022(007)).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.
Horticulture Research
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T2T reference genome assembly provides insights into anthocyanin accumulation in broccoli
21-Apr-2026
The authors declare that they have no competing interests.