A high-quality Bougainvillea genome provides new insights into evolutionary history and pigment biosynthetic pathways in the Caryophyllales

Abstract

Bougainvillea is a perennial ornamental shrub that is highly regarded in ornamental horticulture around the world. However, the absence of genome data limits our understanding of the pathways involved in bract coloration and breeding. Here, we report a chromosome-level assembly of the giga-genome of Bougainvillea × buttiana ‘Mrs Butt’, a cultivar thought to be the origin of many other Bougainvillea cultivars. The assembled genome is ∼5 Gb with a scaffold N50 of 151 756 278 bp and contains 86 572 genes which have undergone recent whole-genome duplication. We confirmed that multiple rounds of whole-genome multiplication have occurred in the evolutionary history of the Caryophyllales, reconstructed the relationship in the Caryophyllales at whole genome level, and found discordance between species and gene trees as the result of complex introgression events. We investigated betalain and anthocyanin biosynthetic pathways and found instances of independent evolutionary innovations in the nine different Caryophyllales species. To explore the potential formation mechanism of diverse bract colors in Bougainvillea, we analyzed the genes involved in betalain and anthocyanin biosynthesis and found extremely low expression of ANS and DFR genes in all cultivars, which may limit anthocyanin biosynthesis. Our findings indicate that the expression pattern of the betalain biosynthetic pathway did not directly correlate with bract color, and a higher expression level in the betalain biosynthetic pathway is required for colored bracts. This improved understanding of the correlation between gene expression and bract color allows plant breeding outcomes to be predicted with greater certainty.

Acknowledgements

We thank Mr Zhihui Xia for valuable comments and helpful discussions. This research was supported by Nanfan special project, CAAS (grant no. SWAQ06), the Funding of Major Scientific Research Tasks, Kunpeng Institute of Modern Agriculture at Foshan (KIMA-ZDKY2022004), Key R&D Project of Hainan Province (ZDYF2022XDNY254), the Scientific Research Foundation for Principle Investigator, Kunpeng Institute of Modern Agriculture at Foshan (KIMA-QD2022004), the Chinese Academy of Agricultural Sciences Elite Youth Program (110243160001007) to Z.W., Science Technology and Innovation Commission of Shenzhen Municipality of China (ZDSYS20200811142605017), and the National Natural Science Foundation of China (32071786).

Author contributions

Z.W. and J.Y. designed the whole of the research; L.L. and X.L. performed the genome assemble and genome annotation. H.Z. and S.X. performed the whole-genome multiplication and karyotype analysis. L.L., S.K., and X.Z. performed the phylogenetic analysis. L.L., H.Z., S.X., W.L., and L.R.T. performed the pigment pathway analysis. L.L. and H.Z. wrote the first draft of the manuscript. L.L., H.Z., S.X., S.K., X.Z., W.L., X.L., L.R.T., Y.R., W.R., J.Y., and Z.W. revised the manuscript, which all authors edited and approved.

Data availability

The genome assembly sequences, gene annotations and transcriptome data are publicly available in the China National GeneBank (https://www.cngb.org/) under project number CNP0004115.

Conflict of interest

The authors declare that they have no competing interests.

References

[1] Abarca-Vargas R, Petricevich VL . Bougainvillea genus: a review on phytochemistry, pharmacology, and toxicology . Evid Based Complement Alternat Med. 2018; 2018: 9070927.

[2] Andrews, S. FastQC: a quality control tool for high throughput sequence data. Babraham Bioinformatics, Babraham Institute, Cambridge, UK, 2010.

[3] Badouin HJ, Gouzy CJ, Grassa F et al. The sunflower genome provides insights into oil metabolism, flowering and asterid evolution. Nature. 2017; 546: 148-52.

[4] Besnard G, Rubio de Casas R, Vargas P . Plastid and nuclear DNA polymorphism reveals historical processes of isolation and reticulation in the olive tree complex (Olea europaea). J Biogeogr. 2007; 34: 736-52.

[5] Brockington SF, Alexandre R, Ramdial J et al. Phylogeny of the Caryophyllales sensu lato: revisiting hypotheses on pollination biology and perianth differentiation in the core Caryophyllales. Int J Plant Sci. 2009; 170: 627-43.

[6] Brockington SF, Yang Y, Gandia-Herrero F et al. Lineage-specific gene radiations underlie the evolution of novel betalain pigmentation in Caryophyllales. New Phytol. 2015; 207: 1170-80.

[7] Bromham L, Hua X, Lanfear R et al. Exploring the relationships between mutation rates, life history, genome size, environment, and species richness in flowering plants. Am Nat. 2015; 185: 507-24.

[8] Brown CT, Irber L . Sourmash: a library for MinHash sketching of DNA. J Open Source Softw. 2016; 1: 27.

[9] Caballero M, Wegrzyn J . gFACs: gene filtering, analysis, and conversion to unify genome annotations across alignment and gene prediction frameworks. Genomics Proteomics Bioinformatics. 2019; 17: 305-10.

[10] Cai X, Sun X, Xu C et al. Genomic analyses provide insights into spinach domestication and the genetic basis of agronomic traits. Nat Commun. 2021; 12: 7246.

[11] Camacho C, Coulouris G, Avagyan V et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009; 10: 421.

[12] Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T . trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009; 25: 1972-3.

[13] Chen L, Qiu Q, Jiang Y et al. Large-scale ruminant genome sequencing provides insights into their evolution and distinct traits. Science. 2019; 364: eaav6202.

[14] Chen Y-A, Lin C-C, Wang C-D et al. An optimized procedure greatly improves EST vector contamination removal. BMC Genomics. 2007; 8: 1-11.

[15] Cheng H, Concepcion GT, Feng X et al. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18: 170-5.

[16] Cuénoud P, Savolainen V, Chatrou LW et al. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Bot. 2002; 89: 132-44.

[17] Gupta YC, Panwar S, Banyal N et al. Floriculture and Ornamental Plants. Singapore: Springer; 2022.

[18] De La Torre AR, Li Z, Van de Peer Y et al. Contrasting rates of molecular evolution and patterns of selection among gymnosperms and flowering plants. Mol Biol Evol. 2017; 34: 1363-77.

[19] Dohm JC, Minoche AE, Holtgräwe D et al. The genome of the recently domesticated crop plant sugar beet (Beta vulgaris). Nature. 2014; 505: 546-9.

[20] Dong W, Liu Y, Li E et al. Phylogenomics and biogeography of catalpa (Bignoniaceae) reveal incomplete lineage sorting and three dispersal events. Mol Phylogenet Evol. 2022; 166: 107330.

[21] Dudchenko O, Batra SS, Omer AD et al. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds . Science. 2017; 356: 92-5.

[22] Durand NC, Shamim MS, Machol I et al. Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Syst. 2016; 3: 95-8.

[23] Edelman NB, Frandsen PB, Miyagi M et al. Genomic architecture and introgression shape a butterfly radiation. Science. 2019; 366: 594-9.

[24] Emms DM, Kelly S . OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019; 20: 1-14.

[25] Enrico E. The French-Italian public consortium for grapevine genome characterization the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nat Cell Biol. 2007; 449: 463-7.

[26] Fawad SA, Khalid N, Asghar W et al. In vitro comparative study of Bougainvillea spectabilis ‘stand’ leaves and Bougainvillea variegata leaves in terms of phytochemicals and antimicrobial activity . Chin J Nat Med. 2012; 10: 441-7.

[27] Flynn JM, Hubley R, Goubert C et al. RepeatModeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci USA. 2020; 117: 9451-7.

[28] Freeling M, Lyons E, Pedersen B et al. Many or most genes in Arabidopsis transposed after the origin of the order Brassicales . Genome Res. 2008; 18: 1924-37.

[29] Gandía-Herrero F, Cabanes J, Escribano J et al. Encapsulation of the most potent antioxidant betalains in edible matrixes as powders of different colors. J Agric Food Chem. 2013; 61: 4294-302.

[30] Gandía-Herrero F, García-Carmona F . Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci. 2013; 18: 334-43.

[31] Gilman IS, Moreno-Villena JJ, Lewis ZR et al. Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca. Plant Physiol. 2022; 189: 735-53.

[32] Chase MW, Christenhusz, Fay MJM, MF et al. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc. 2016; 181: 1-20.

[33] Gu Z, Eils R, Schlesner M . Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics. 2016; 32: 2847-9.

[34] Guo X, Fang D, Sahu SK et al. Chloranthus genome provides insights into the early diversification of angiosperms . Nat Commun. 2021; 12: 6930.

[35] Haas BJ, Delcher AL, Mount SM et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies . Nucleic Acids Res. 2003; 31: 5654-66.

[36] Haas BJ, Papanicolaou A, Yassour M et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013; 8: 1494-512.

[37] Haas BJ, Salzberg SL, Zhu W et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 2008; 9: R7-22.

[38] Hanada K, Zou C, Lehti-Shiu MD et al. Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli. Plant Physiol. 2008; 148: 993-1003.

[39] Hernández-Ledesma P, Berendsohn WG, Borsch T et al. A taxonomic backbone for the global synthesis of species diversity in the angiosperm order Caryophyllales. Willdenowia. 2015; 45: 281-383.

[40] Hoff KJ, Lomsadze A, Borodovsky M et al. Whole-genome annotation with BRAKER. Methods Mol Biol. 2019; 1962: 65-95.

[41] Huang X, Huang S, Han B et al. The integrated genomics of crop domestication and breeding. Cell. 2022; 185: 2828-39.

[42] Huson DH, Scornavacca C . Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol. 2012; 61: 1061-7.

[43] Jain G, Gould KS . Are betalain pigments the functional homologues of anthocyanins in plants? Environ Exp Bot. 2015; 119: 48-53.

[44] Jones P, Binns D, Chang H-Y et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014; 30: 1236-40.

[45] Julca I, Marcet-Houben M, Vargas P et al. Phylogenomics of the olive tree (Olea europaea) reveals the relative contribution of ancient allo-and autopolyploidization events . BMC Biol. 2018; 16: 1-15.

[46] Kang X, Mikami R, Akita Y . Characterization of 5-O-glucosyltransferase involved in anthocyanin biosynthesis in Cyclamen purpurascens. Plant Biotechnol. 2021; 38: 263-8.

[47] Kang Y, Lee H, Kim M-K et al. The complete chloroplast genome of Antarctic pearlwort, Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae). Mitochondrial DNA. 2016; 27: 4677-8.

[48] Katoh K, Standley DM . MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772-80.

[49] Kumar S, Stecher G, Suleski M et al. TimeTree: a resource for timelines, timetrees, and divergence times. Mol Biol Evol. 2017; 34: 1812-9.

[50] Leebens-Mack JH, Barker MS, Carpenter EJ et al. One thousand plant transcriptomes and the phylogenomics of green plants. Nature. 2019; 574: 679-85.

[51] Li F, Gao Y, Jin C et al. The chromosome-level genome of Gypsophila paniculata reveals the molecular mechanism of floral development and ethylene insensitivity . Hortic Res. 2022; 9: uhac176.

[52] Li H . Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34: 3094-100.

[53] Li H, Niu J, Sun L et al. Ploidy levels and DNA contents of Bougainvillea accessions determined by flow cytometry analysis . Hort Sci. 2022; 57: 1502-6.

[54] 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.

[55] Lynch M, Conery JS . The evolutionary fate and consequences of duplicate genes. Science. 2000; 290: 1151-5.

[56] Ma X, Vaistij FE, Li Y et al. A chromosome-level Amaranthus cruentus genome assembly highlights gene family evolution and biosynthetic gene clusters that may underpin the nutritional value of this traditional crop . Plant J. 2021; 107: 613-28.

[57] Mackintosh A, Vila R, Laetsch DR et al. Chromosome fissions and fusions act as barriers to gene flow between Brenthis fritillary butterflies . Mol Biol Evol. 2023; 40: msad043.

[58] Maddison WP . Gene trees in species trees. Syst Biol. 1997; 46: 523-36.

[59] Mai U, Mirarab S . TreeShrink: fast and accurate detection of outlier long branches in collections of phylogenetic trees. BMC Genomics. 2018; 19: 23-40.

[60] Manni M, Berkeley MR, Seppey M et al. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021; 38: 4647-54.

[61] Mapleson D, Garcia Accinelli G, Kettleborough G et al. KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies. Bioinformatics. 2017; 33: 574-6.

[62] Mistry J, Bateman A, Finn RD . Predicting active site residue annotations in the Pfam database. BMC Bioinformatics. 2007; 8: 298.

[63] Mistry J, Finn RD, Eddy SR et al. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 2013; 41:e121-1.

[64] Nguyen L-T, Schmidt HA, Von Haeseler A et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32: 268-74.

[65] Ou S, Su W, Liao Y et al. Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline. Genome Biol. 2019; 20: 275.

[66] Parins-Fukuchi C, Stull GW, Smith SA . Phylogenomic conflict coincides with rapid morphological innovation. Proc Natl Acad Sci USA. 2021; 118: e2023058118.

[67] Paterson AH, Freeling M, Tang H et al. Insights from the comparison of plant genome sequences. Annu Rev Plant Biol. 2010; 61: 349-72.

[68] Pellicer J, Hidalgo O, Dodsworth S et al. Genome size diversity and its impact on the evolution of land plants. Genes. 2018; 9: 88.

[69] Qiao X, Li Q, Yin H et al. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019; 20: 38.

[70] Rambaut A, Drummond AJ, Xie D et al. Posterior summarization in Bayesian phylogenetics using tracer 1.7. Syst Biol. 2018; 67: 901-4.

[71] Salam P, Bhargav V, Gupta Y et al. Evolution in bougainvillea (Bougainvillea Commers.) - a review . J Appl Nat Sci. 2017; 9: 1489-94.

[72] Sayers EW, Beck J, Bolton EE et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2021; 49: D1-10.

[73] Smith SA, Brown JW, Yang Y et al. Disparity, diversity, and duplications in the Caryophyllales. New Phytol. 2018; 217: 836-54.

[74] Smith SA, Moore MJ, Brown JW et al. Analysis of phylogenomic datasets reveals conflict, concordance, and gene duplications with examples from animals and plants. BMC Evol Biol. 2015; 15: 150.

[75] Smith SA, Walker-Hale N, Walker JF et al. Phylogenetic conflicts, combinability, and deep phylogenomics in plants. Syst Biol. 2020; 69: 579-92.

[76] Soltis DE, Albert VA, Leebens-Mack J et al. Polyploidy and angiosperm diversification. Am J Bot. 2009; 96: 336-48.

[77] Standley PC . The nyctaginaceae and chenopodiaceae of northwestern South America. In: Field Museum of Natural History-Botanical Series, Vol. 11. Chicago, IL, USA, 1931, 73-114.

[78] Stanke M, Keller O, Gunduz I et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006; 34: W435-9.

[79] Stanke M, Schöffmann O, Morgenstern B et al. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics. 2006; 7: 62.

[80] Stull GW, Pham KK, Soltis PS et al. Deep reticulation: the long legacy of hybridization in vascular plant evolution. Plant J. 2022; 114: 743-66.

[81] Sun H, Ding J, Piednoël M et al. findGSE: estimating genome size variation within human and Arabidopsis using k-mer frequencies . Bioinformatics. 2018; 34: 550-7.

[82] Sun P, Jiao B, Yang Y et al. WGDI: a user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. Mol Plant. 2022; 15: 1841-51.

[83] Suvorov A, Kim BY, Wang J et al. Widespread introgression across a phylogeny of 155 Drosophila genomes . Curr Biol. 2022; 32: 111-123.e5.

[84] Tanaka Y, Sasaki N, Ohmiya A . Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54: 733-49.

[85] Tossi VE, Tosar LM, Pitta-Álvarez SI et al. Casting light on the pathway to betalain biosynthesis: a review. Environ Exp Bot. 2021; 186: 104464.

[86] Walker JF, Yang Y, Moore MJ et al. Widespread paleopolyploidy, gene tree conflict, and recalcitrant relationships among the carnivorous Caryophyllales. Am J Bot. 2017; 104: 858-67.

[87] Wang M, Zhang L, Tong S et al. Chromosome-level genome assembly of a xerophytic plant, Haloxylon ammodendron. DNA Res. 2022; 29: dsac006.

[88] Wang P, Wang F . A proposed metric set for evaluation of genome assembly quality. Trends Genet. 2023; 39: 175-86.

[89] Wen D, Yu Y, Zhu J et al. Inferring phylogenetic networks using PhyloNet. Syst Biol. 2018; 67: 735-40.

[90] Datta SK, Jayanthi R, Bougainvillea JT . Bougainvillaeas. Floric Ornam Plants. 2020: 1-34.

[91] Wu Q, Fu X, Chen Z et al. Composition, color stability and antioxidant properties of betalain-based extracts from bracts of Bougainvillea. Molecules. 2022; 27: 5120.

[92] Xiong X, Gou J, Liao Q et al. The Taxus genome provides insights into paclitaxel biosynthesis . Nature Plants. 2021; 7: 1026-36.

[93] Yang J, Huang J, Gu H et al. Duplication and adaptive evolution of the chalcone synthase genes of Dendranthema (Asteraceae). Mol Biol Evol. 2002; 19: 1752-9.

[94] Yang Z . PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24: 1586-91.

[95] Yao G, Jin J-J, Li H-T et al. Plastid phylogenomic insights into the evolution of Caryophyllales. Mol Phylogenet Evol. 2019; 134: 74-86.

[96] Yi L, Sa R, Zhao S et al. Chromosome-scale, haplotype-resolved genome assembly of Suaeda glauca. Front Genet. 2022; 13: 884081.

[97] Zhang B-W, Xu L-L, Li N et al. Phylogenomics reveals an ancient hybrid origin of the Persian walnut. Mol Biol Evol. 2019; 36: 2451-61.

[98] Zhang C, Rabiee M, Sayyari E et al. ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinformatics. 2018; 19: 15-30.

[99] Zhang C, Zhang T, Luebert F et al. Asterid phylogenomics/phylotranscriptomics uncover morphological evolutionary histories and support phylogenetic placement for numerous whole-genome duplications. Mol Biol Evol. 2020; 37: 3188-210.

[100] Zhang X, Lin S, Peng D et al. Integrated multi-omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers. Plant Biotechnol J. 2022; 20: 1182-96.