A telomere-to-telomere gap-free genome of the new cultivar ‘Zhongtian No. 5’, combined with pan-genome analysis, aids in exploration and genetic enhancement of red clover ( Trifolium pratense L.)

Abstract

Trifolium pratense L. is a multifunctional crop of agronomic importance for forage, horticulture, and ecological restoration. However, the lack of a high-quality genome assembly and the limited representation of genetic diversity by a single reference have impeded its genetic research and molecular breeding. Here, we present the first telomere-to-telomere (T2T) gap-free genome for the diploid (2n = 2 x = 14) cultivarT. pratense cv. ‘Zhongtian No. 5’ (TpraZt5), assembled through an integrated sequencing strategy. The 390.94 Mb assembly demonstrates high quality, with a base accuracy > 98.5%, 98.1% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness, a long terminal repeat assembly index of 25.65, and a contig N50 of 52.95 Mb. We annotated 35 971 protein-coding genes and found repeat sequences accounting for 59.6% of the genome. The assembly resolved all seven centromeres and 14 telomeres, providing unprecedented insight into these complex genomic regions. We further constructed a 480.76 Mb pan-genome by integrating two additional accessions, which classified genes into core (70.2%), dispensable (25.3%), and private (4.5%) sets. Comparative genomic analyses identified 606 species-specific genes in TpraZt5 and uncovered extensive structural variations. Functional investigations revealed four species-specific genes and six contracted genes associated with isoflavonoid biosynthesis, two expanded chlorophyll a–b-binding proteins, and seven expanded auxin-related genes that may contribute to the high productivity of TpraZt5. Additionally, 44 Gypsy-type transposons within the zeatin biosynthesis pathway were identified as potential regulators of trifoliate leaf development. These genomic resources substantially improve structural annotation and functional characterization, providing vital tools for gene discovery and enhancing molecular breeding initiatives in red clover.

Acknowledgements

This research was funded by National Natural Science Foundation of China (32401292), the Youth Innovation Program of Chinese Academy of Agricultural Sciences (Y2023QC31), Lanzhou Young Scientific & Technological Talent Program (2024-QN-4), the Natural Science Foundation of Gansu Province (25JRRA452, 22JR5RA040), Fundamental Research Funds for the Central Public-interest Scientific Institution (1610322025004), and the Science and Technology Innovation Program of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences (CAAS-LMY-04).

Ethics approval and consent to participate

No ethical approval or permission is required to obtain the materials and perform the research in this study.

Author contributions

G.C., H.D., and H.Y. conceived and designed the research. G.C., J.Z., T.G., and F.W. collected the samples. G.C. performed the genome assembly and conducted the experimental and data analysis. F.W. prepared the figures. X.W., B.S., Xi.Z., Q.Z., Y.L., and Xu.Z. participated in data analysis. G.C. wrote the manuscript. H.D. submitted the data. H.Y., H.D., and C.W. revised the manuscript.

Data availability

The genome sequencing data, including PacBio HiFi, ONT Ultra-long, and Hi-C data, have been deposited into the NCBI database and are available via the BioProject accession number PRJNA1332894. All additional supporting data are available in the supplementary materials.

Conflicts of interest statement

The authors declare no conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Riday H . Progress made in improving red clover (Trifolium pratense L.) through breeding . Int J Plant Breed. 2010; 4: 22-9

[2] Broderick GA . Utilization of protein in red clover and alfalfa silages by lactating dairy cows and growing lambs. J Dairy Sci. 2018; 101: 1190-205

[3] Stefan A , Van Cauwenberghe J , Rosu CM , et al. Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables . Syst Appl Microbiol. 2018; 41: 251-9

[4] Albrecht KA, Muck RE . Proteolysis in ensiled forage legumes that vary in tannin concentration. Crop Sci. 1991; 31: 464-9

[5] Oleszek W, Stochmal A, Janda B . Concentration of isoflavones and other phenolics in the aerial parts of Trifolium species . J Agric Food Chem. 2007; 55: 8095-100

[6] Kolodziejczyk-Czepas J, Krzyzanowska-Kowalczyk J, Sieradzka M, et al. Clovamide and clovamide-rich extracts of three Trifolium species as antioxidants and moderate antiplatelet agents in vitro . Phytochemistry. 2017; 143: 54-63

[7] Riday H, Krohn AL . Genetic map-based location of the red clover (Trifolium pratense L.) gametophytic self-incompatibility locus . Theor Appl Genet. 2010; 121: 761-7

[8] Ištvanek J, Jaroš M, Křenek A, et al. Genome assembly and annotation for red clover (Trifolium pratense; Fabaceae) . Am J Bot. 2014; 101: 327-37

[9] Naish M, Alonge M, Wlodzimierz P, et al. The genetic and epigenetic landscape of the Arabidopsis centromeres. Science. 2021; 374: eabi7489

[10] Song JM, Xie WZ, Wang S, et al. Two gap-free reference genomes and a global view of the centromere architecture in rice. Mol Plant. 2021; 14: 1757-67

[11] Wang LF, Zhang MZ, Li MN, et al. A telomere-to-telomere gap-free assembly of soybean genome. Mol Plant. 2023; 16: 1711-4

[12] Zhang C, Xie L, Yu H, et al. The T2T genome assembly of soybean cultivar ZH13 and its epigenetic landscapes. Mol Plant. 2023; 16: 1715-8

[13] Gong S, Xie W, Zhao R, et al. Progress and prospect of plant telomere-to-telomere (T2T) genome. Genomics Appl Biol . 2024; 43: 933-42 (in Chinese)

[14] Bayer PE, Golicz AA, Scheben A, et al. Plant pan-genomes are the new reference. Nat Plants. 2020; 6: 914-20

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

[16] Ou L, Li D, Lv J, et al. Pan-genome of cultivated pepper (capsicum) and its use in gene presence-absence variation analyses. New Phytol. 2018; 220: 360-3

[17] Sun X, Jiao C, Schwaninger H, et al. Phased diploid genome assemblies and pan-genomes provide insights into the genetic history of apple domestication. Nat Genet. 2020; 52: 1423-32

[18] Liu Y, Du H, Li P, et al. Pan-genome of wild and cultivated soybeans. Cell. 2020; 182: 162-176.e13

[19] Hu G, Cheng L, Cheng Y, et al. Pan-genome analysis of three main Chinese chestnut varieties. Front Plant Sci . 2022; 13: 916550

[20] 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

[21] Zhao Q, Feng Q, Lu H, et al. Pan-genome analysis highlights the extent of genomic variation in cultivated and wild rice. Nat Genet. 2018; 50: 278-84

[22] Shi J, Tian Z, Lai J, et al. Plant pan-genomics and its applications. Mol Plant. 2023; 16: 168-86

[23] Wang M, Li J, Qi Z, et al. Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium. Nat Genet. 2022; 54: 1959-71

[24] Ni L, Liu Y, Ma X, et al. Pan-3D genome analysis reveals structural and functional differentiation of soybean genomes. Genome Biol . 2023; 24: 12

[25] Murukarthick J, Mona S, Nils S, et al. Building pan-genome infrastructures for crop plants and their use in association genetics. DNA Res. 2021; 28: dsaa030

[26] Bickhart DM, Koch LM, Smith TPL, et al. Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species . GigaByte. 2022; 2022: 1-13

[27] Hirakawa H, Kaur P, Shirasawa K, et al. Draft genome sequence of subterranean clover, a reference for genus Trifolium . Sci Rep. 2016; 6: 30358

[28] De Vega JJ, Ayling S, Hegarty M, et al. Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement . Sci Rep. 2015; 5: 17394

[29] Yan ZF, Sang LJ, Ma Y, et al. A de novo assembled high-quality chromosome-scale Trifolium pratense genome and fine-scale phylogenetic analysis. BMC Plant Biol. 2022; 22: 332

[30] Xia Y, Ning Z, Bai G, et al. Allelic variations of a light harvesting chlorophyll a/b binding protein gene (Lhcb1) associated with agronomic traits in barley . PLoS One. 2012; 7: e37573

[31] Luo J, Abid M, Tu J, et al. Genome-wide identification of the LHC gene family in kiwifruit and regulatory role of AcLhcb3.1/3.2 for chlorophyll a content . Int J Mol Sci. 2022; 23: 6528

[32] Ma Q, Li H, Zou Z, et al. Transcriptomic analyses identify albino-associated genes of a novel albino tea germplasm ’Huabai 1’. Hortic Res. 2018; 5: 54

[33] Lin W, Guo X, Pan X, et al. Chlorophyll composition, chlorophyll fluorescence, and grain yield change in esl mutant rice . Int J Mol Sci. 2018; 19: 2945

[34] Neoh BK, Wong YC, Teh HF, et al. Diurnal biomarkers reveal key photosynthetic genes associated with increased oil palm yield. PLoS One. 2019; 14: e0213591

[35] Wu P, Kong Q, Bian J, et al. Unveiling molecular mechanisms of nitric oxide-induced low-temperature tolerance in cucumber by transcriptome profiling. Int J Mol Sci. 2022; 23: 5615

[36] Zhao S, Gao H, Luo J, et al. Genome-wide analysis of the light-harvesting chlorophyll a/b binding gene family in apple (Malus domestica) and functional characterization of MdLhcb4.3, which confers tolerance to drought and osmotic stress . Plant Physiol Biochem. 2020; 154: 517-29

[37] Huang J, Zhao X, Chory J . The Arabidopsis transcriptome responds specifically and dynamically to high light stress . Cell Rep. 2019; 29: 4186-99

[38] Jin JH, Zhang HX, Tan JY, et al. A new ethylene-responsive factor CaPTI1 gene of pepper (Capsicum annuum L.) involved in the regulation of defense response to Phytophthora capsici . Front Plant Sci. 2016; 6: 1217

[39] Bao D, Chang S, Li X, et al. Advances in the study of auxin early response genes: Aux/IAA, GH3, and SAUR . Crop J. 2024; 12: 964-78

[40] Dubey SM, Han S, Stutzman N, et al. The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana . Mol Plant. 2023; 16: 1120-30

[41] Farcot E, Lavedrine C, Vernoux T . A modular analysis of the auxin signalling network. PLoS One. 2015; 10: e0122231

[42] Devoghalaere F, Doucen T, Guitton B, et al. A genomics approach to understanding the role of auxin in apple (Malus × domestica) fruit size control . BMC Plant Biol. 2012; 12: 7

[43] Zhang ZY . Cloning and Functional Mechanism Analysis of Rice Yield Gene Gnp4 . Beijing: China Agricultural University; 2015

[44] Ding YF, Zeng WF, Wang XB, et al. Over-expression of peach PpIAA19 in tomato alters plant growth, parthenocarpy, and fruit shape . J Plant Growth Regul. 2019; 38: 103-12

[45] Ren H, Gray WM . SAUR proteins as effectors of hormonal and environmental signals in plant growth. Mol Plant. 2015; 8: 1153-64

[46] Stamm P, Kumar PP . Auxin and gibberellin responsive Arabidopsis SMALL AUXIN UP RNA36 regulates hypocotyl elongation in the light. Plant Cell Rep. 2013; 32: 759-69

[47] Wang JJ, Sun N, Zhang FF, et al. SAUR17 and SAUR50 differentially regulate PP2C-D1 during apical hook development and cotyledon opening in Arabidopsis. Plant Cell . 2020; 32: 3792-811

[48] Li YH, Zhou G, Ma J, et al. De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits. Nat Biotechnol. 2014; 32: 1045-52

[49] Zhou Y, Zhang Z, Bao Z, et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature. 2022; 606: 527-34

[50] Kaur H, Shannon LM, Samac DA . A stepwise guide for pangenome development in crop plants: an alfalfa (Medicago sativa) case study . BMC Genomics. 2024; 25: 1022

[51] Knox AK, Dhillon T, Cheng H, et al. CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals. Theor Appl Genet. 2010; 121: 21-35

[52] Maron LG, Guimarães CT, Kirst M, et al. Aluminum tolerance in maize is associated with higher MATE1 gene copy number. Proc Natl Acad Sci USA. 2013; 110: 5241-6

[53] Cook DE, Lee TG, Guo X, et al. Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean. Science. 2012; 338: 1206-9

[54] Sutton T, Baumann U, Hayes J, et al. Boron-toxicity tolerance in barley arising from efflux transporter amplification. Science. 2007; 318: 1446-9

[55] Nitcher R, Distelfeld A, Tan C, et al. Increased copy number at the HvFT1 locus is associated with accelerated flowering time in barley. Mol Gen Genomics. 2013; 288: 261-75

[56] Lin Z, Li X, Shannon LM, et al. Parallel domestication of the Shattering1 genes in cereals. Nat Genet. 2012; 44: 720-4

[57] Zhou Y, Zhu J, Li Z, et al. Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication. Genetics. 2009; 183: 315-24

[58] Studer A, Zhao Q, Ross-Ibarra J, et al. Identification of a functional transposon insertion in the maize domestication gene tb1. Nat Genet. 2011; 43: 1160-3

[59] Yan H, Sun M, Zhang Z, et al. Pangenomic analysis identifies structural variation associated with heat tolerance in pearl millet. Nat Genet. 2023; 55: 507-18

[60] Malaviya DR, Roy AK, Kaushal P, et al. Phenotype study of multifoliolate leaf formation in Trifolium alexandrinum L. Peer J. 2021; 9: e10874

[61] Song IJ, Kang HG, Kang JY, et al. Breeding of four-leaf white clover (Trifolium repens L.) through 60Co gamma-ray irradiation. Plant Biotechnol Rep. 2009; 3: 191-7

[62] Vasanthi RP . Inheritance of pentafoliate character in groundnut (Arachis hypogaea L.) . Indian J Plant Genet Resour. 2003; 16: 79-84

[63] Soehendi R, Chanprame S, Toojinda T, et al. Genetics, agronomic, and molecular study of leaflet mutants in mungbean (Vigna radiata (L.) Wilczek) . J Crop Sci Biotechnol. 2007; 10: 193-200

[64] Chatterton NJ . Photosynthesis of 22 alfalfa populations differing in resistance to diseases, insect, pests and nematodes. Crop Sci. 1976; 16: 833-4

[65] Kaur A, Kaur KP, Kalia A, et al. Generation of interspecific hybrids between Trifolium vesiculosum and T. alexandrinum using embryo rescue. Euphytica. 2017; 213: 253

[66] Juan NA, Sheaffer CC, Barnes DK, et al. Leaf and stem traits and herbage quality of multifoliolate alfalfa. Agron J. 1993; 85: 1121-7

[67] Ellison NW, Liston A, Steiner JJ, et al. Molecular phylogenetics of the clover genus (Trifolium-Leguminosae). Mol Phylogenet Evol. 2006; 39: 688-705

[68] Simon U . Inheritance of polyphylly in red clover (Trifolium pratense L.) . Crop Sci. 1962; 2: 258

[69] Jaranowski JK, Broda Z . Leaf mutants in diploid red clover (Trifolium pratense L.) . Theor Appl Genet. 1978; 53: 97-103

[70] Taylor NL . Registration of gene marker germplasm for redclover. Crop Sci . 1982; 22: 1269

[71] Zotz G, Wilhelm K, Becker A . Heteroblasty-a review. Bot Rev . 2011; 77: 109-51

[72] Rose KME, Mickelbart MV, Jacobs DF . Plasticity of phenotype and heteroblasty in contrasting populations of Acacia koa. Ann Bot . 2019; 124: 399-409

[73] Yan J, Ma C, Bo C, et al. A modified CTAB method for genomic DNA extraction from apple fruit. Mol Plant Breed. 2018; 9: 3610-5

[74] Mario S , Oliver K , Irfan G , et al. Ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006; 34: 435-9

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

[76] 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

[77] 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

[78] Jain C, Rhie A, Zhang HW, et al. Weighted minimizer sampling improves long read mapping. Bioinformatics . 2020; 36: 111-8

[79] Vaser R, Sović I, Nagarajan N, et al. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 2017; 27: 737-46

[80] Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-60

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

[82] Ou S, Jiang N . LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 2018; 176: 1410-22

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

[84] Price AL, Jones NC, Pevzner PA . De novo identification of repeat families in large genomes. Bioinformatics. 2005; 21: 351-8

[85] Zhang HY, He Q, Xing LS, et al. The haplotype-resolved genome assembly of autotetraploid rhubarb Rheum officinale provides insights into its genome evolution and massive accumulation of anthraquinones. Plant Commun. 2024; 5: 100677

[86] Chen N . Using RepeatMasker to identify repetitive elements in genomic sequences. Current Protoc Bioinformatics. 2009; 4: 4.10.1-4.10.14

[87] Jurka J, Kapitonov VV, Pavlicek A, et al. Repbase update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 2005; 110: 462-7

[88] Xu Z, Wang H . LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 2007; 35: W265-8

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

[90] He Q, Li W, Miao YQ, et al. The near-complete genome assembly of hexaploid wild oat reveals its genome evolution and divergence with cultivated oats. Nat Plants. 2024; 10: 2062-78

[91] Majoros WH, Pertea M, Salzberg SL . TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics . 2004; 20: 2878-9

[92] 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

[93] Holt C, Yandell M . MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011; 12: 491

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

[95] Edgar RC . MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32: 1792-7

[96] Stamatakis A . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30: 1312-3

[97] Hedges SB, Dudley J, Kumar S . TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006; 22: 2971-2

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

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

[100] Yang Z . PAML: a program package for phylogenetic analysis by maximum likelihood. Bioinformatics. 1997; 13: 555-6

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

[102] Ul Qamar MT, Zhu X, Xing F, et al. PpsPCP: a plant presence/absence variants scanner and pan-genome construction pipeline. Bioinformatics. 2019; 35: 4156-8

[103] Mi H, Muruganujan A, Ebert D, et al. PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res. 2019; 47: D419-26

[104] Yu G, Wang LG, Han Y, et al. ClusterProfiler: an R package for comparing biological themes among gene clusters. Omics. 2012; 16: 284-7

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