Complete telomere-to-telomere genomes of cowpea reveal insights into centromere evolution in Phaseoleae

Abstract

Cowpea ( Vigna unguiculata) is a versatile legume crop providing a critical source of grain, vegetable and forage globally. Cultivated cowpea is classified into two main subspecies, subsp. sesquipedalis for fresh-pod vegetable and subsp. unguiculata for grain production. Here, we present two complete telomere-to-telomere (T2T) assemblies for the grain-type inbred lines HJD and vegetable-type FC6 through integrating PacBio HiFi reads, Oxford Nanopore ultralong reads, and Hi-C data. The T2T genomes demonstrated improved contiguity, completeness, and accuracy compared to existing genomes, revealing clear telomeric and centromeric features. Comparative analysis of the T2T genomes highlighted inversions underlying subspecies divergence in cowpea. Evolutionary analysis uncovered contraction of gene families related to symbiosis in HJD, consist with its reduced root nodules compared to FC6. Distribution and composition of tandem repeat arrays and transposable elements in centromeric regions were largely conserved in cowpea, but displayed pronounced variation among Phaseoleae. Furthermore, frequent shifts of centromeric locations coincided with inversions found in Phaseoleae. Overall, this study provides a set of fundamental resources for cowpea improvement and enhances our understanding of cowpea subspecies divergence and genome evolution in Phaseoleae.

Acknowledgements

This work was financially supported by State Key Laboratory for Tropical Crop Breeding and the start-up package from Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences.

Author contributions

Y.F.T. and Y.Y. conceived the project. C.Z.W., S.C.S., Y.Z.W., and S.P. performed bioinformatic analyses. Y.Z.W., Y.B.W., and T.D. conducted plant cultivation and experiments. C.Z.W. and Y.F.T. wrote the manuscript. E.M. and D.J. supervised the work. All authors contributed to the article and approved the submitted version.

Data availability

Raw sequencing reads and assembly results for HJD have been deposited under project PRJCA044301 at the National Genomics Data Center. Protein-coding gene annotation files and additional assembly resources have been uploaded to the online repository Figshare (URL: https://figshare.com/articles/dataset/cowpea_genome_project/29878346). The scripts used for the analysis are available in a GitHub repository at https://github.com/ChuanzhengWei/cowpea_T2T.

Conflicts of interest statement

The authors declared that they have no conflict of interest in relation to this work.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Ehlers JD, Hall AE . Cowpea (Vigna unguiculata L. Walp.). Field Crop Res. 1997; 53: 187-204

[2] Chen H, Chen H, Hu L, et al. Genetic diversity and a population structure analysis of accessions in the Chinese cowpea [Vigna unguiculata (L.) Walp.] germplasm collection. Crop J. 2017; 5: 363-72

[3] Chipeta MM, Kafwambira J, Yohane E . Cowpea genetic diversity, population structure and genome-wide association studies in Malawi: insights for breeding programs. Front Plant Sci. 2025; 15: 1461631

[4] Boukar O, Fatokun CA, Huynh B-L, et al. Genomic tools in cowpea breeding programs: status and perspectives. Front Plant Sci. 2016; 7: 757

[5] Pasquet RS . Allozyme diversity of cultivated cowpea Vigna unguiculata (L.) Walp. Theor Appl Genet. 2000; 101: 211-9

[6] Kouakou A-G, Ogundapo A, Smale M, et al. IITA’s genebank, cowpea diversity on farms, and farmers’ welfare in Nigeria. CABI Agric Biosci. 2022; 3: 14

[7] Fang J, Chao C-CT, Roberts PA, et al. Genetic diversity of cowpea [Vigna unguiculata (L.) Walp.] in four West African and USA breeding programs as determined by AFLP analysis. Genet Resour Crop Evol. 2007; 54: 1197-209

[8] Xu P, Wu X, Wang B, et al. Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.). Mol Breed. 2010; 25: 675-84

[9] Jayathilake C, Visvanathan R, Deen A, et al. Cowpea: an overview on its nutritional facts and health benefits. J Sci Food Agric. 2018; 98: 4793-806

[10] Affrifah NS, Phillips RD, Saalia FK . Cowpeas: nutritional profile, processing methods and products-a review. Legume Sci. 2022; 4: e131

[11] Della Coletta R, Qiu Y, Ou S, et al. How the pan-genome is changing crop genomics and improvement. Genome Biol. 2021; 22: 3

[12] Muñoz-Amatriaín M, Mirebrahim H, Xu P, et al. Genome resources for climate-resilient cowpea, an essential crop for food security. Plant J. 2017; 89: 1042-54

[13] Lonardi S, Muñoz-Amatriaín M, Liang Q, et al. The genome of cowpea (Vigna unguiculata [L.] Walp.). Plant J. 2019; 98: 767-82

[14] Xia Q, Pan L, Zhang R, et al. The genome assembly of asparagus bean, Vigna unguiculata ssp sesquipedalis. Sci Data. 2019; 6: 124

[15] Yang Y, Wu Z, Wu Z, et al. A near-complete assembly of asparagus bean provides insights into anthocyanin accumulation in pods. Plant Biotechnol J. 2023; 21: 2473-89

[16] Wang X, Zhou P, Hu X, et al. T2T genome, pan-genome analysis, and heat stress response genes in Rhododendron species. iMeta. 2025; 4: e70010

[17] Gao W, Wang S, Jiang T, et al. Chromosome-scale and haplotype-resolved genome assembly of Populus trichocarpa. Hortic Res. 2025; 12: 012

[18] Li Q, Qiao X, Li L, et al. Haplotype-resolved T2T genome assemblies and pangenome graph of pear reveal diverse patterns of allele-specific expression and the genomic basis of fruit quality traits. Plant Commun. 2024; 5: 101000

[19] Kajita T, Ohashi H, Tateishi Y, et al. rbcL and legume phylogeny, with particular reference to Phaseoleae, Millettieae, and allies. Syst Bot. 2001; 26: 515-36

[20] Lackey JA . Chromosome numbers in the Phaseoleae (Fabaceae: Faboideae) and their relation to taxonomy. Am J Bot. 1980; 67: 595-602

[21] Vasconcelos EV, de Andrade Fonsêca AF, Pedrosa-Harand A, et al. Intra- and interchromosomal rearrangements between cowpea [Vigna unguiculata (L.) Walp.] and common bean (Phaseolus vulgaris L.) revealed by BAC-FISH. Chromosom Res. 2015; 23: 253-66

[22] de Oliveira Bustamante F, do Nascimento TH, Montenegro C, et al. Oligo-FISH barcode in beans: a new chromosome identification system. Theor Appl Genet. 2021; 134: 3675-86

[23] Oliveira AR, Martins L, Bustamante F, et al. Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris). Chromosom Res. 2020; 28: 293-306

[24] Cleveland DW, Mao Y, Sullivan KF . Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell. 2003; 112: 407-21

[25] do Vale Martins L, de Oliveira Bustamante F, da Silva Oliveira AR, et al. BAC- and oligo-FISH mapping reveals chromosome evolution among Vigna angularis, V. unguiculata, and Phaseolus vulgaris. Chromosoma. 2021; 130: 133-47

[26] Montenegro C, do Vale Martins L, Bustamante F, et al. Comparative cytogenomics reveals genome reshuffling and centromere repositioning in the legume tribe Phaseoleae. Chromosom Res. 2022; 30: 477-92

[27] Wang L, Zhang M, Li M, et al. A telomere-to-telomere gap-free assembly of soybean genome. Mol Plant. 2023; 16: 1711-4

[28] Wang Y, Hao X, Chen C, et al. Telomere-to-telomere genome of common bean (Phaseolus vulgaris L., YP4). GigaScience. 2025; 14: giaf001

[29] Zhao B, Zhang H, Zhao Q, et al. Gap-free genome assembly and metabolomics analysis of common bean provide insights into genomic characteristics and metabolic determinants of seed coat pigmentation. J Genet Genomics. 2025; 52: 852-5

[30] Jia K-H, 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

[31] Liu J, Li Q, Hu Y, et al. The complete telomere-to-telomere sequence of a mouse genome. Science. 2024; 386: 1141-6

[32] Naish M, Henderson IR . The structure, function, and evolution of plant centromeres. Genome Res. 2024; 34: 161-78

[33] Pan L, Liu M, Kang Y, et al. Comprehensive genomic analyses of Vigna unguiculata provide insights into population differentiation and the genetic basis of key agricultural traits. Plant Biotechnol J. 2023; 21: 1426-39

[34] Wellenreuther M, Bernatchez L . Eco-evolutionary genomics of chromosomal inversions. Trends Ecol Evol. 2018; 33: 427-40

[35] Wang J, Sun P, Li Y, et al. Hierarchically aligning 10 legume genomes establishes a family-level genomics platform. Plant Physiol. 2017; 174: 284-300

[36] Schmutz J, Cannon SB, Schlueter J, et al. Genome sequence of the palaeopolyploid soybean. Nature. 2010; 463: 178-83

[37] Young ND, Debellé F, Oldroyd GED, et al. The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature. 2011; 480: 520-4

[38] Liu Y-Y, Yu C-L, Liu Y-J, et al. An autotetraploid genome of Corydalis sheareri provides insight into the evolution and benzylisoquinoline alkaloids diversity of Corydalis. Genomics Communications. 2025; 2: e002

[39] Yang Y, Du W, Li Y, et al. Recent advances and challenges in de novo genome assembly. Genomics Communications. 2025; 2: e104

[40] Bilgrav Saether K , Eisfeldt J , Bengtsson JD , et al. Leveraging the T2T assembly to resolve rare and pathogenic inversions in reference genome gaps. Genome Res. 2024; 34: 1785-97

[41] Shang L, He W, Wang T, et al. A complete assembly of the rice Nipponbare reference genome. Mol Plant. 2023; 16: 1232-6

[42] Chen J, Wang Z, Tan K, et al. A complete telomere-to-telomere assembly of the maize genome. Nat Genet. 2023; 55: 1221-31

[43] Wei C, Gao L, Xiao R, et al. Complete telomere-to-telomere assemblies of two sorghum genomes to guide biological discovery. iMeta. 2024; 3: e193

[44] Kumon T, Lampson MA . Evolution of eukaryotic centromeres by drive and suppression of selfish genetic elements. Semin Cell Dev Biol. 2022; 128: 51-60

[45] Hu G, Wang Z, Tian Z, et al. A telomere-to-telomere genome assembly of cotton provides insights into centromere evolution and short-season adaptation. Nat Genet. 2025; 57: 1031-43

[46] Jin X, Du H, Chen M, et al. A fully phased octoploid strawberry genome reveals the evolutionary dynamism of centromeric satellites. Genome Biol. 2025; 26: 17

[47] Courret C, Hemmer LW, Wei X, et al. Turnover of retroelements and satellite DNA drives centromere reorganization over short evolutionary timescales in Drosophila. PLoS Biol. 2024; 22: e3002911

[48] Zhao J, Xie Y, Kong C, et al. Centromere repositioning and shifts in wheat evolution. Plant Commun. 2023; 4: 100556

[49] Wang Y, Zhao L, Wang D, et al. Four near-complete genome assemblies reveal the landscape and evolution of centromeres in Salicaceae. Genome Biol. 2025; 26: 111

[50] Huang G, Wang X, Liu C, et al. Genomic variation underpins genetic divergence and differing salt resilience in Sesbania bispinosa. Adv Sci. 2025; 12: e02600

[51] Blumer LM, Burskaia V, Artiushin I, et al. Introgression dynamics of sex-linked chromosomal inversions shape the Malawi cichlid radiation. Science. 2025; 388: eadr9961

[52] Ahmed HI, Heuberger M, Schoen A, et al. Einkorn genomics sheds light on history of the oldest domesticated wheat. Nature. 2023; 620: 830-8

[53] Zhang T, Bao Y, You H, et al. T2T assembly of Oryza australiensis reveals centromere repositioning mechanisms and identifies the EE genome as direct donor of the extinct DD lineage. Research Square Preprint. 2025. https://doi.org/10.21203/rs.3.rs-7517070/v1

[54] Porubsky D, Sanders AD, Höps W, et al. Recurrent inversion toggling and great ape genome evolution. Nat Genet. 2020; 52: 849-58

[55] Francis BA, Gozashti L, Costello K, et al. Complete genome assemblies of two mouse subspecies reveal structural diversity of telomeres and centromeres. Nat Genet. 2025; 57: 2852-62

[56] Porebski S, Bailey LG, Baum BR . Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Report. 1997; 15: 8-15

[57] Rao SSP, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014; 159: 1665-80

[58] Marçais G, Kingsford C . A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27: 764-70

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

[60] Cheng H, Jarvis ED, Fedrigo O, et al. Haplotype-resolved assembly of diploid genomes without parental data. Nat Biotechnol. 2022; 40: 1332-5

[61] Li H . Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. 2013; arXiv:1303.3997

[62] Zeng X, Yi Z, Zhang X, et al. Chromosome-level scaffolding of haplotype-resolved assemblies using Hi-C data without reference genomes. Nat Plants. 2024; 10: 1184-200

[63] Rautiainen M, Nurk S, Walenz BP, et al. Telomere-to-telomere assembly of diploid chromosomes with Verkko. Nat Biotechnol. 2023; 41: 1474-82

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

[65] Jain C, Rhie A, Hansen NF, et al. Long-read mapping to repetitive reference sequences using Winnowmap2. Nat Meth. 2022; 19: 705-10

[66] 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

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

[68] Yu H, Shi C, He W, et al. PanDepth, an ultrafast and efficient genomic tool for coverage calculation. Brief Bioinform. 2024; 25: bbae197

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

[70] Hu K, Ni P, Xu M, et al. HiTE: a fast and accurate dynamic boundary adjustment approach for full-length transposable element detection and annotation. Nat Commun. 2024; 15: 5573

[71] Zhang R-G, Li G-Y, Wang X-L, et al. TEsorter: an accurate and fast method to classify LTR-retrotransposons in plant genomes. Hortic Res. 2022; 9: uhac017

[72] Iserić H, Alkan C, Hach F, et al. Fast characterization of segmental duplication structure in multiple genome assemblies. Algorithms Mol Biol. 2022; 17: 4

[73] Kim D, Paggi JM, Park C, et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019; 37: 907-15

[74] Gabriel L, Brůna T, Hoff KJ, et al. BRAKER3: fully automated genome annotation using RNA-seq and protein evidence with GeneMark-ETP, AUGUSTUS, and TSEBRA. Genome Res. 2024; 34: 769-77

[75] 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

[76] 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

[77] Lan L, Hu H, Jia Y, et al. Tips for improving genome annotation quality. Genomics Communications. 2025; 2: e005

[78] Buchfink B, Xie C, Huson DH . Fast and sensitive protein alignment using DIAMOND. Nat Meth. 2015; 12: 59-60

[79] Lin Y, Ye C, Li X, et al. quarTeT: a telomere-to-telomere toolkit for gap-free genome assembly and centromeric repeat identification. Hortic Res. 2023; 10: uhad127

[80] Wlodzimierz P , Hong M , Henderson IR . TRASH: tandem repeat annotation and structural hierarchy. Bioinformatics. 2023; 39: btad308

[81] Vollger MR, Kerpedjiev P, Phillippy AM, et al. StainedGlass: interactive visualization of massive tandem repeat structures with identity heatmaps. Bioinformatics. 2022; 38: 2049-51

[82] Van der Auwera GA , Carneiro MO , Hartl C , et al. From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr Protoc Bioinformatics. 2013; 43: 11.10.1-11.10.33

[83] Chang CC, Chow CC, Tellier LCAM, et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience. 2015; 4: s13742-015-0047-8

[84] Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol. 2011; 29: 24-6

[85] Goel M, Sun H, Jiao W-B, et al. SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20: 277

[86] Goel M, Schneeberger K . Plotsr: visualizing structural similarities and rearrangements between multiple genomes. Bioinformatics. 2022; 38: 2922-6

[87] Emms DM, Kelly S . OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019; 20: 238

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

[89] Mendes FK, Vanderpool D, Fulton B, et al. CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics. 2021; 36: 5516-8

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

[91] Tang H, Krishnakumar V, Zeng X, et al. JCVI: a versatile toolkit for comparative genomics analysis. iMeta. 2024; 3: e211

[92] Chen H, Van Zwaenepoel A, de Peer Y . Wgd v2: a suite of tools to uncover and date ancient polyploidy and whole-genome duplication. Bioinformatics. 2024; 40: btae272

[93] Xie J, Chen Y, Cai G, et al. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res. 2023; 51: W587-92

[94] He W, Yang J, Jing Y, et al. NGenomeSyn: an easy-to-use and flexible tool for publication-ready visualization of syntenic relationships across multiple genomes. Bioinformatics. 2023; 39: btad121