A nearly complete haplotype-phased genome assembly of nerve plant (Fittonia albivenis) provides insights into leaf color evolution

Abstract

Fittonia albivenis, commonly known as the nerve plant, is an ornamental species native to the Peruvian rainforest, valued for its vibrant and diverse leaf coloration. Understanding the genetic mechanisms underlying this coloration is crucial for enhancing its ornamental value and adaptation to environmental stressors. Here, by leveraging advanced sequencing technologies such as PacBio HiFi, Oxford Nanopore, and Hi-C, we achieved a nearly complete haplotype-phased genome assembly for F. albivenis, revealing a 2.08-Gb genome composed of 18 chromosome pairs and containing 66 telomeres. This assembly enabled the identification of subgenome-specific repetitive sequences, elucidating their impact on gene expression and structural variations. Through RNA sequencing, metabolomic profiling, and resequencing, we dissected the regulatory networks influencing chlorophyll and anthocyanin biosynthesis, identifying key genes and transcription factors driving leaf color variation. Our findings highlight the roles of gene duplication and specific transcription factors in pigment synthesis pathways, providing a foundation for future genetic studies and breeding programs aimed at enhancing ornamental and adaptive traits in F. albivenis and related species.

Acknowledgments

The project was funded by the National Natural Science Foundation of China (32270260, 32070267), Shandong Provincial Natural Science Foundation, China (ZR2019ZD48) and Taishan Scholars Project (X.Q.).

Author contributions

X.Q. conceived and designed the study. L.W., R.G.Z., and K.H.J. conducted experiments and analyzed data. L.W. and K.H.J. wrote the manuscript. X.Q. and K.H.J. edited and improved the manuscript. All authors approved the final manuscript.

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA017781) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.The whole-genome sequence data reported in this paper have been deposited in the Genome Warehouse in National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences/China National Center for Bioinformation, under accession number of GWHEUVN00000000.1 that is publicly accessible at https://ngdc.cncb.ac.cn/gwh.

Conflict of interest statement

The authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Zhao M-H, Li X, Zhang X-X. et al. Mutation mechanism of leaf color in plants: a review. Forests. 2020; 11:851

[2] Liu Y, Feng X, Zhang Y. et al. Simultaneous changes in antho-cyanin, chlorophyll, and carotenoid contents produce green variegation in pink-leaved ornamental kale. BMC Genomics. 2021; 22:455

[3] Zhu PK, Zeng MY, Lin YH. et al. Variability in leaf color induced by chlorophyll deficiency: transcriptional changes in bamboo leaves. Curr Issues Mol Biol. 2024; 46:1503-15

[4] Valenta K, Dimac-Stohl K, Baines F. et al. Ultraviolet radiation changes plant color. BMC Plant Biol. 2020; 20:253

[5] Mathesius U. Flavonoid functions in plants and their interactions with other organisms. Plants (Basel). 2018; 7:30

[6] Guo S, Zhang M, Feng M. et al. miR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought. Hortic Res. 2024;11:uhad293

[7] Jia KH, Wang ZX, Wang L. et al. SubPhaser: a robust allopolyploid subgenome phasing method based on subgenome-specific k-mers. New Phytol. 2022; 235:801-9

[8] Zhang Z, Chen C, Jiang C. et al. VvWRKY5 positively regulates wounding-induced anthocyanin accumulation in grape by inter-playing with VvMYBA1 and promoting jasmonic acid biosynthe-sis. Hortic Res. 2024;11:uhae083

[9] Li G, Tang L, He Y. et al. The haplotype-resolved T2T reference genome highlights structural variation underlying agronomic traits of melon. Hortic Res. 2023;10:uhad182

[10] Jia KH, Zhang X, Li LL. et al. Telomere-to-telomere genome assemblies of cultivated and wild soybean provide insights into evolution and domestication under structural variation. Plant Commun. 2024; 5:100919

[11] Jia KH, Li G, Wang L. et al. Telomere-to-telomere, gap-free genome of mung bean (Vigna radiata) provides insights into domestication under structural variation. Hortic Res. 2025;12: uhae337

[12] Shi TL, Jia KH, Bao YT. et al. High-quality genome assembly enables prediction of allele-specific gene expression in hybrid poplar. Plant Physiol. 2024; 195:652-70

[13] Ranallo-Benavidez TR, Jaron KS, Schatz MC. GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat Commun. 2020; 11:1432

[14] 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

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

[16] Robinson JT, Turner D, Durand NC. et al. Juicebox.Js provides a cloud-based visualization system for Hi-C data. Cell Systems. 2018; 6:256-258.e1

[17] Hu J, Wang Z, Sun Z. et al. NextDenovo: an efficient error cor-rection and accurate assembly tool for noisy long reads. Genome Biol. 2024; 25:107

[18] Jin JJ, Yu WB, Yang JB. et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020; 21:241

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

[20] Chen N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2004;Chapter 4: Unit 4 10

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

[22] Pertea M, Pertea GM, Antonescu CM. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015; 33:290-5

[23] Simão FA, Waterhouse RM, Ioannidis P. et al. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31:3210-2

[24] Rhie A, Walenz BP, Koren S. et al. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 2020; 21:245

[25] Zhang RG, Shang HY, Milne RI. et al. SOI: robust identifica-tion of orthologous synteny with the Orthology Index and broad applications in evolutionary genomics. Nucleic Acids Res. 2025;53:gkaf320

[26] Zhang RG, Li GY, Wang XL. et al. TEsorter: an accurate and fast method to classify LTR-retrotransposons in plant genomes. Hortic Res. 2022;9:uhac017

[27] Wang WB, He XF, Yan XM. et al. Chromosome-scale genome assembly and insights into the metabolome and gene regulation of leaf color transition in an important oak species, Quercus dentata. New Phytol. 2023; 238:2016-32

[28] Yang FS, Nie S, Liu H. et al. Chromosome-level genome assembly of a parent species of widely cultivated azaleas. Nat Commun. 2020; 11:5269

[29] Zeng T, He Z, He J. et al. The telomere-to-telomere gap-free reference genome of wild blueberry (Vaccinium duclouxii)provides its high soluble sugar and anthocyanin accumulation. Hortic Res. 2023;10:uhad209

[30] Zhang K, du M, Zhang H. et al. The haplotype-resolved T2T genome of teinturier cultivar Yan73 reveals the genetic basis of anthocyanin biosynthesis in grapes. Hortic Res. 2023;10:uhad205

[31] Conant GC, Wolfe KH. Turning a hobby into a job: how duplicated genes find new functions. Nat Rev Genet. 2008; 9:938-50

[32] Bekaert M, Edger PP, Pires JC. et al.Two-phase resolution of polyploidy in the Arabidopsis metabolic network gives rise to relative and absolute dosage constraints. Plant Cell. 2011; 23: 1719-28

[33] Thevenot EA, Roux A, Xu Y. et al. Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for uni-variate and OPLS statistical analyses. JProteomeRes. 2015; 14: 3322-35

[34] Lloyd A, Brockman A, Aguirre L. et al. Advances in the MYB-bHLH-WD repeat (MBW) pigment regulatory model: addition of a WRKY factor and co-option of an antho-cyanin MYB for betalain regulation. Plant Cell Physiol. 2017; 58: 1431-41

[35] Yao G, Ming M, Allan AC. et al. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant J. 2017; 92:437-51

[36] Vimolmangkang S, Han Y, Wei G. et al. An apple MYB tran-scription factor, MdMYB3, is involved in regulation of antho-cyanin biosynthesis and flower development. BMC Plant Biol. 2013; 13:176

[37] Long F, Wu H, Li H. et al. Genome-wide analysis of MYB tran-scription factors and screening of MYBs involved in the red color formation in Rhododendron delavayi. Int J Mol Sci. 2023; 24: 4641

[38] Rushton PJ, Somssich IE, Ringler P. et al. WRKY transcription factors. Trends Plant Sci. 2010; 15:247-58

[39] Martins TR, Berg JJ, Blinka S. et al. Precise spatio-temporal regula-tion of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae). New Phytol. 2013; 197: 958-69

[40] Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987; 19:11-5

[41] Wang C, Liu C, Roqueiro D. et al. Genome-wide analysis of local chromatin packing in Arabidopsis thaliana. Genome Res. 2015; 25: 246-56

[42] 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

[43] Hu J, Fan J, Sun Z. et al. NextPolish: a fast and efficient genome polishing tool for long-read assembly. Bioinformatics. 2020; 36: 2253-5

[44] Pryszcz LP, Gabaldon T. Redundans: an assembly pipeline for highly heterozygous genomes. Nucleic Acids Res. 2016; 44:e113

[45] Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27:573-80

[46] Gel B, Serra E. karyoploteR: an R/Bioconductor package to plot customizable genomes displaying arbitrary data. Bioinformatics. 2017; 33:3088-90

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

[48] Stanke M, Diekhans M, Baertsch R. et al. Using native and syn-tenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008; 24:637-44

[49] Cantarel BL, Korf I, Robb SMC. et al. MAKER: an easy-to-use anno-tation pipeline designed for emerging model organism genomes. Genome Res. 2008; 18:188-96

[50] 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

[51] Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detec-tion of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-64

[52] Kalvari I, Argasinska J, Quinones-Olvera N. et al. Rfam 13.0: shift-ing to a genome-centric resource for non-coding RNA families. Nucleic Acids Res. 2018;46:D335-42

[53] Cantalapiedra CP, Hernández-Plaza A, Letunic I. et al. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021; 38:5825-9

[54] Buchfink B, Xie C, Huson DH. Fast and sensitive protein align-ment using DIAMOND. Nat Methods. 2015; 12:59-60

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

[56] Goel M, Sun H, Jiao WB. et al. SyRI: finding genomic rear-rangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20:277

[57] Goel M, Schneeberger K. Plotsr: visualizing structural similar-ities and rearrangements between multiple genomes. Bioinfor-matics. 2022; 38:2922-6

[58] Zhang RG, Shang HY, Jia KH. et al. Subgenome phasing for complex allopolyploidy: case-based benchmarking and recom-mendations. Brief Bioinform. 2023;25:bbad513

[59] Zhang RG, Lu C, Li GY. et al.Subgenome-aware analyses suggest a reticulate allopolyploidization origin in three Papaver genomes. Nat Commun. 2023; 14:2204

[60] Qiao X, Li Q, Yin H. et al. Gene duplication and evolution in recur-ring polyploidization-diploidization cycles in plants. Genome Biol. 2019; 20:1-23

[61] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:238

[62] Yin J, Zhang C, Mirarab S. ASTRAL-MP: scaling ASTRAL to very large datasets using randomization and parallelization. Bioinfor-matics. 2019; 35:3961-9

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

[64] De Bie T, Cristianini N, Demuth JP. et al. CAFE: a computa-tional tool for the study of gene family evolution. Bioinformatics. 2006; 22:1269-71

[65] Chen S, Zhou Y, Chen Y. et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884-90

[66] Patro R, Duggal G, Love MI. et al. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14:417-9

[67] Soneson C, Love MI, Robinson MD. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res. 2015; 4:1521

[68] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550

[69] Chen W, Gong L, Guo Z. et al. A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics. Mol Plant. 2013; 6:1769-80

[70] Zhu G, Wang S, Huang Z. et al. Rewiring of the fruit metabolome in tomato breeding. Cell. 2018; 172:249-261.e12

[71] Tian F, Yang DC, Meng YQ. et al. PlantRegMap: chart-ing functional regulatory maps in plants. Nucleic Acids Res. 2020;48:D1104-13

[72] JinJ, TianF, YangDC. et al. PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res. 2017;45:D1040-5

[73] Shannon P, Markiel A, Ozier O. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13:2498-504

[74] Garrison E,Marth G. Haplotype-based variant detection from short-read sequencingarxiv preprint arxiv:1207.3907. 2012

[75] Danecek P, Auton A, Abecasis G. et al. The variant call format and VCFtools. Bioinformatics. 2011; 27:2156-8

[76] Narasimhan V, Danecek P, Scally A. et al. BCFtools/RoH: a hidden Markov model approach for detecting autozygosity from next-generation sequencing data. Bioinformatics. 2016; 32:1749-51

[77] Purcell S, Neale B, Todd-Brown K. et al. PLINK: a tool set for whole-genome association and population-based linkage anal-yses. Am J Hum Genet. 2007; 81:559-75

[78] Alexander DH, Novembre J, Lange K. Fast model-based estima-tion of ancestry in unrelated individuals. Genome Res. 2009; 19: 1655-64

[79] Yang J, Lee SH, Goddard ME. et al. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011; 88:76-82

[80] Kopelman NM, Mayzel J, Jakobsson M. et al. Clumpak: a pro-gram for identifying clustering modes and packaging popu-lation structure inferences across K. Mol Ecol Resour. 2015; 15: 1179-91

[81] Chen JH, Huang Y, Brachi B. et al. Genome-wide analysis of Cushion willow provides insights into alpine plant divergence in a biodiversity hotspot. Nat Commun. 2019; 10:5230

[82] Wu T, Hu E, Xu S. et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb). 2021; 2:100141

[83] Yu G. Enrichplot: visualization of functional enrichment resultR package version 1. 2021;