T2T reference genome assembly provides insights into anthocyanin accumulation in broccoli

Abstract

Broccoli (Brassica oleracea var. italica) is a widely cultivated cruciferous vegetable valued for its abundant bioactive compounds and nutraceutical properties. Among these, anthocyanins are not only important secondary metabolites contributing to nutritional and medicinal benefits, but also influence stress tolerance and the commercial quality of broccoli through purple pigmentation. However, the molecular mechanisms regulating anthocyanin biosynthesis in broccoli remain poorly understood, partly due to the incomplete genomic resources currently available. In this study, we constructed a telomere-to-telomere gap-free assembly of the broccoli genome using a combination of Oxford Nanopore Technology ultralong reads, PacBio high-fidelity reads, and Hi-C datasets. The resulting genome is 633.61 Mb in length, with an N50 of 60.36 Mb, and comprises gap-free assemblies of all 18 chromosomes, including complete telomere-to-telomere assemblies for nine chromosomes. Using this high-quality reference, we identified BoF3’H, a key gene regulating anthocyanin accumulation, which controlling the purple coloration of broccoli buds. To validate the function of the BoF3’H gene in anthocyanin biosynthesis, we used CRISPR-Cas9 gene editing to target and knock out the BoF3’H gene. The bof3’h mutant exhibited an 81.4% reduction in cyanidin and delphinidin derivative levels compared with those in the control. Metabolomic and transcriptomic profiling showed that the expression of 12 anthocyanin-related genes, including PAL, C4H, CL3, CHS, F3’H, and ANS, was downregulated. These findings elucidate the molecular basis of anthocyanin regulation in broccoli and provide a foundational genomic resource for evolutionary studies, gene discovery, and future breeding.

References

[1] Kan S, Liao X, Lan L, et al. Cytonuclear interactions and subgenome dominance shape the evolution of organelle-targeted genes in the brassica triangle of U. Mol Biol Evol. 2024; 41: msae043

[2] Yan L, Zhou G, Shahzad K, et al. Research progress on the utilization technology of broccoli stalk, leaf resources, and the mechanism of action of its bioactive substances. Front Plant Sci. 2023; 14: 1138700

[3] Li H, Xia Y, Liu HY, et al. Nutritional values, beneficial effects, and food applications of broccoli (Brassica oleracea var. italica Plenck). Trends Food Sci Technol. 2022; 119: 288-308

[4] Parkin IA, Koh C, Tang H, et al. Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol. 2014; 15: 1-18

[5] Liu S, Liu Y, Yang X, et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun. 2014; 5: 3930

[6] Cai X, Wu J, Liang J, et al. Improved Brassica oleracea JZS assembly reveals significant changing of LTR-RT dynamics in different morphotypes. Theor Appl Genet. 2020; 133: 3187-99

[7] Chen R, Chen K, Yao X, et al. Genomic analyses reveal the stepwise domestication and genetic mechanism of curd biogenesis in cauliflower. Nat Genet. 2024; 56: 1235-44

[8] Guo N, Wang S, Gao L, et al. Genome sequencing sheds light on the contribution of structural variants to Brassica oleracea diversification. BMC Biol. 2021; 19: 93

[9] Lv H, Wang Y, Han F, et al. A high-quality reference genome for cabbage obtained with SMRT reveals novel genomic features and evolutionary characteristics. Sci Rep. 2020; 10: 12394

[10] Sun D, Wang C, Zhang X, et al. Draft genome sequence of cauliflower (Brassica oleracea L. var. botrytis) provides new insights into the C genome in Brassica species. Hortic Res. 2019; 6: 82

[11] Wu Q, Mao S, Huang H, et al. Chromosome-scale reference genome of broccoli (Brassica oleracea var. italica Plenck) provides insights into glucosinolate biosynthesis. Hortic Res. 2024; 11: uhae063

[12] Li H, Durbin R . Genome assembly in the telomere-to-telomere era. Nat Rev Genet. 2024; 25: 658-70

[13] Garg V, Bohra A, Mascher M, et al. Unlocking plant genetics with telomere-to-telomere genome assemblies. Nat Genet. 2024; 56: 1788-99

[14] Wang YH, Liu PZ, Liu H, et al. Telomere-to-telomere carrot (Daucus carota) genome assembly reveals carotenoid characteristics. Hortic Res. 2023; 10: uhad103

[15] Zhou Y, Xiong J, Shu Z, et al. The telomere-to-telomere genome of Fragaria vesca reveals the genomic evolution of Fragaria and the origin of cultivated octoploid strawberry. Hortic Res. 2023; 10: uhad027

[16] Li F, Gong Y, Mason AS, et al. Research progress and applications of colorful Brassica crops. Planta. 2023; 258: 45

[17] Liu CQ, Yao XQ, Li GQ, et al. Transcriptomic profiling of purple broccoli reveals light-induced anthocyanin biosynthetic signaling and structural genes. PeerJ. 2020; 8: e8870

[18] Yu H, Wang J, Shen Y, et al. A 43 Bp-deletion in the F3′H gene reducing anthocyanins is responsible for keeping buds green at low temperatures in broccoli. Int J Mol Sci. 2023; 24: 11391

[19] Tanaka Y, Sasaki N, Ohmiya A . Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2018; 54: 733-49

[20] Wen S, Li N, Song S, et al. Comparative transcriptome and metabolome analyses of broccoli germplasms with purple and green curds reveal the structural genes and transitional regulators regulating color formation. Int J Mol Sci. 2023; 24: 6115

[21] Liu CQ, Yao XQ, Li GQ, et al. Development of novel markers and creation of non-anthocyanin and anthocyanin-rich broccoli (Brassica oleracea var. italica) cultivars. Appl Sci. 2022; 12: 6267

[22] Khusnutdinov E, Sukhareva A, Panfilova M, et al. Anthocyanin biosynthesis genes as model genes for genome editing in plants. Int J Mol Sci. 2021; 22: 8752

[23] Zhai Y, Yu K, Cai S, et al. Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L. Plant Biotechnol J. 2020; 18: 1153-68

[24] Ren J, Liu Z, Niu R, et al. Mapping of Re, a gene conferring the red leaf trait in ornamental kale (Brassica oleracea L. var. acephala). Plant Breed. 2015; 134: 494-500

[25] Zhang X, Zhang K, Wu J, et al. QTL-Seq and sequence assembly rapidly mapped the gene BrMYBL2.1 for the purple trait in Brassica rapa. Sci Rep. 2020; 10: 2328

[26] Liu CQ, Yao XQ, Li GQ, et al. Identification of major loci and candidate genes for anthocyanin biosynthesis in broccoli using QTL-Seq. Horticulturae. 2021; 7: 246

[27] Cabello-Hurtado F, El Amrani A . Phenanthrene-induced cytochrome P450 genes and phenanthrene tolerance associated with Arabidopsis thaliana CYP75B1 gene. Plants. 2024; 13: 1692

[28] Park S, Lee H, Min MK, et al. Functional characterization of BrF3’H, which determines the typical flavonoid profile of purple Chinese cabbage. Front Microbiol. 2021; 12: 793589

[29] Li S, Li X, Wang X, et al. Flavonoid synthesis-related genes determine the color of flower petals in Brassica napus L. Int J Mol Sci. 2023; 24: 6472

[30] Li X, Wang Y, Cai C, et al. Large-scale gene expression alterations introduced by structural variation drive morphotype diversification in Brassica oleracea. Nat Genet. 2024; 56: 517-29

[31] He Z, Ji R, Havlickova L, et al. Genome structural evolution in Brassica crops. Nat Plants. 2021; 7: 757-65

[32] Xu L, Wang Y, Dong J, et al. A chromosome-level genome assembly of radish (Raphanus sativus L.) reveals insights into genome adaptation and differential bolting regulation. Plant Biotechnol J. 2023; 21: 990-1004

[33] Zhang L, Liang J, Chen H, et al. A near-complete genome assembly of Brassica rapa provides new insights into the evolution of centromeres. Plant Biotechnol J. 2023; 21: 1022-32

[34] Belser C, Istace B, Denis E, et al. Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps. Nat Plants. 2018; 4: 879-87

[35] Zhang Y, Yang Z, He Y, et al. Structural variation reshapes population gene expression and trait variation in 2,105 Brassica napus accessions. Nat Genet. 2024; 56: 2538-50

[36] Guo N, Cheng F, Wu J, et al. Anthocyanin biosynthetic genes in Brassica rapa. BMC Genomics. 2014; 15: 1-11

[37] Tang K, Karamat U, Li G, et al. Integrated metabolome and transcriptome analyses reveal the role of BoGSTF12 in anthocyanin accumulation in Chinese kale (Brassica oleracea var. alboglabra). BMC Plant Biol. 2024; 24: 335

[38] He Q, Zhang Z, Zhang L . Anthocyanin accumulation, antioxidant ability and stability, and a transcriptional analysis of anthocyanin biosynthesis in purple heading Chinese cabbage (Brassica rapa L. ssp. pekinensis). J Agric Food Chem. 2016; 64: 132-45

[39] Yu H, Wang J, Zhao Z, et al. Construction of a high-density genetic map and identification of loci related to hollow stem trait in broccoli (Brassica oleracea L. italica). Front Plant Sci. 2019; 10: 45

[40] Adachi T, Okamoto H, Shiroto Y, et al. Identification of a candidate gene and development of a marker for anthocyanin-free florets at low temperature in Japanese broccoli (Brassica oleracea var. italica). Hort J. 2025; 94: 232

[41] Jung YJ, Lee HJ, Kim JH, et al. CRISPR/Cas9-targeted mutagenesis of F3′H, DFR and LDOX, genes related to anthocyanin biosynthesis in black rice (Oryza sativa L.). Plant Biotechnol Rep. 2019; 13: 521-31

[42] Marçais G, Delcher AL, Phillippy AM, et al. MUMmer4: a fast and versatile genome alignment system. PLoS Comput Biol. 2018; 14: e1005944

[43] Love MI, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 1-21