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A telomere-to-telomere genome assembly of cotton provides insights into centromere evolution and short-season adaptation

Nature Genetics volume 57, pages 1031–1043 (2025)Cite this article

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Abstract

Cotton (Gossypium hirsutum L.) is a key allopolyploid crop with global economic importance. Here we present a telomere-to-telomere assembly of the elite variety Zhongmian 113. Leveraging technologies including PacBio HiFi, Oxford Nanopore Technology (ONT) ultralong-read sequencing and Hi-C, our assembly surpasses previous genomes in contiguity and completeness, resolving 26 centromeric and 52 telomeric regions, 5S rDNA clusters and nucleolar organizer regions. A phylogenetically recent centromere repositioning on chromosome D08 was discovered specific to G. hirsutum, involving deactivation of an ancestral centromere and the formation of a unique, satellite repeat-based centromere. Genomic analyses evaluated favorable allele aggregation for key agronomic traits and uncovered an early-maturing haplotype derived from an 11 Mb pericentric inversion that evolved early during G. hirsutum domestication. Our study sheds light on the genomic origins of short-season adaptation, potentially involving introgression of an inversion from primitively domesticated forms, followed by subsequent haplotype differentiation in modern breeding programs.

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Fig. 1: T2T assembly and validation of ZM113 v1.0.
Fig. 2: Characteristics of ZM113 centromeric regions.
Fig. 3: Evolutionary dynamics of the D08 centromere.
Fig. 4: Structural and GCVs in G. hirsutum.
Fig. 5: Identification of an INV-derived haplotype associated with FD on chromosome D03.

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Data availability

The ZM113 genome assembly and annotation were deposited in NCBI under BioProject accession number PRJNA1137578 and Cotton Breeding Database (accessible at http://222.88.152.130:1130/). The raw sequencing reads by ONT ultralong-read sequencing, PacBio HiFi sequencing, MGI PE150 short-read sequencing, Hi-C, RNA-seq, ChIP-seq and BS-seq are available in NCBI under BioProject PRJNA1041574 and Cotton Breeding Database (accessible at http://222.88.152.130:1130/). The raw Bionano data are available in the Genome Sequence Archive of China National Genomics Data Center under PRJCA029603 accession CRA018637 (accessible at https://ngdc.cncb.ac.cn/gsa/). Nine G. hirsutum reference genomes and two diploid cotton genomes were downloaded from CottonGen (accessible at https://cottongen.org/). Genetic variant VCF for Hap-D03-1 to Hap-D03-5 was deposited in https://github.com/huguanjing/cottonRef_ZM113/. Source data are provided with this paper.

Code availability

Custom scripts are available in the GitHub repository (https://github.com/huguanjing/cottonRef_ZM113) and on Zenodo (https://doi.org/10.5281/zenodo.14840103)127.

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Acknowledgements

This study was supported by the National Key Research and Development Program of China (2021YFF1000100 to Xiongfeng Ma), the National Natural Science Foundation of China (32072114 to Xiongfeng Ma, 32072111 to G.H. and 32070544 to K.W.), the Key Research and Development project of Xinjiang Uygur Autonomous Region (2022B02052-2 to Xiongfeng Ma), the Tianshan Innovation Team of Xinjiang Uygur Autonomous Region (2023D14016 to G.H.), the Major Science and Technology Program of Changji Hui Autonomous Prefecture (2021Z01-01 to Xiongfeng Ma), the Postdoctoral and High-level Flexible Talents of Xinjiang Uygur Autonomous Region (RSSQ00066509 to X. Zhang), the China Agriculture Research System (CARS-15-07 to Xiongfeng Ma), the Shennong Talents program (SNYCQN002-2022 to Xiongfeng Ma), the Top Talent Project of Henan Province (ZYYCYU202012146 to Xiongfeng Ma), the Natural Science Foundation of Henan Province (202300410550 to Xiongfeng Ma) and the Chinese Academy of Agricultural Sciences (the Innovation Project CAAS-ASTIP-ICR-KP-2021-01 to Xiongfeng Ma, the Youth Innovation Project Y2023QC38 to Z. Wang, the Young Elite Scientists Sponsorship Program by Henan Association for Science and Technology (2024HYTP010 to Z.T.) and the Special Project Cotton Research Institute 1610162023005 to X.W.). We thank X. Du (Cotton Research Institute, Chinese Academy of Agricultural Sciences) and J. Zhang (Guangxi University) for discussion and critical reading of the paper.

Author information

Author notes

  1. These authors contributed equally: Guanjing Hu, Zhenyu Wang, Zunzhe Tian, Kai Wang, Gaoxiang Ji, Xingxing Wang, Xianliang Zhang, Zhaoen Yang.

Authors and Affiliations

  1. National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China

    Guanjing Hu, Zhenyu Wang, Gaoxiang Ji, Xingxing Wang, Xianliang Zhang, Zhaoen Yang, Ruoyu Niu, Yuzhi Zhang, Lian Duan, Junjie Zhao, Shoupu He, Xinshan Zang & Xiongfeng Ma

  2. Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Guanjing Hu, Zunzhe Tian, Xuan Liu, Ruoyu Niu, De Zhu, Lian Duan, Xueyuan Ma, Xianpeng Xiong, Jiali Kong, Xianjia Zhao, Ya Zhang, Zhiqiang Wu, Weihua Pan & Xiongfeng Ma

  3. Zhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou, China

    Zhenyu Wang, Gaoxiang Ji, Xingxing Wang, Xianliang Zhang, Zhaoen Yang, Xuan Liu, Junjie Zhao, Shoupu He, Xinshan Zang & Xiongfeng Ma

  4. School of Life Sciences, Nantong University, Nantong, China

    Kai Wang, Guangrun Yu & Jinlei Han

  5. Western Research Institute, Chinese Academy of Agricultural Sciences, Changji, China

    Xianliang Zhang

  6. Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa, USA

    Corrinne E. Grover & Jonathan F. Wendel

  7. State Key Laboratory of Aridland Crop Science, College of Life Science and Technology, Gansu Agricultural University, Lanzhou, China

    Junji Su

  8. Crop Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou, China

    Keyun Feng

Authors
  1. Guanjing Hu
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  2. Zhenyu Wang
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Contributions

Xiongfeng Ma conceived this project and coordinated research activities. G.H. and X.W. designed the experiments. X. Zhang, X.W. and K.F. prepared the plant materials. Z.T., X.W., G.H., X.L., R.N., D.Z., Xueyuan Ma, X.X., Ya Zhang, X. Zang and W.P. assembled, validated and annotated the ZM113 genome. K.F. and Z. Wu assembled and annotated the cytoplasmic genomes. K.W., G.Y. and J.H. performed the ChIP-seq experiment. L.D., Ya Zhang, G.H. and X.X. analyzed the ChIP-seq data. Yuzhi Zhang performed FISH and PCR experiments. Z.T., Ya Zhang and G.H. performed the centromere characterization and evolutionary analysis. X.L. performed the SV analysis. R.N. performed pan-genomic and RNA-seq analyses. Xueyuan Ma analyzed the bisulfite sequencing data. Z. Wang, G.J., Z.Y., J.Z. and X.W. conducted the GWAS and haplotype analysis, with inputs from J.S., S.H. and X. Zang. G.H., Z.T., Z. Wang, G.J. and X.W. wrote the paper, with inputs from D.Z. and X.X. for genome annotation, X.L. and R.N. for pan-genomic analysis, Xueyuan Ma for DNA methylation analysis, and Ya Zhang for ChIP-seq analysis. K.W., C.E.G. and J.F.W. revised the paper. All authors read and approved the paper.

Corresponding author

Correspondence to Xiongfeng Ma.

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The authors declare no competing interests.

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Nature Genetics thanks Yuxian Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Syntenic plot of two different assembly versions of ZM113.

Reference (ref), a previous version assembled using only ONT ultralong with NextDenovo (v2.3.1). Query (qry), the current version assembled using both the ONT ultralong and PacBio HiFi reads. The current assembly is larger in size (2,299.07 Mb vs 2288.35 Mb) and displayed notable improvement on A09 and D09 regions both harboring 5S rDNA clusters.

Extended Data Fig. 2 Potential assembly error (D10) and heterzygous sites (A01, A09, A12, D04 and D08) detected by Veritymap K-mer distance concordance test.

Based on the k-mer discrepancies between HiFi reads and the assembly, if a significant proportion of reads (20-80%) deviate from the assembly at a specific ___location, a heterozygous site is suspected; if nearly all reads deviate (>80%), a potential assembly error is indicated.

Extended Data Fig. 3 Whole-genome alignment between ZM113 and five TM-1 reference genome assemblies.

SyRI visualization of synteny (gray), inversion (orange), translocation (green) and duplication (blue) are shown. Common inversions between ZM113 and TM-1 references are indicated by red arrows.

Extended Data Fig. 4 Collinearity of A08 chromosome between ZM113 and 11 G. hirsutum genome assemblies.

SyRI visualization of synteny (gray), inversion (orange), translocation (green) and duplication (blue) are shown.

Extended Data Fig. 5 Collinearity of D03 chromosome in seven G. hirsutum assemblies.

The newly published TM-1 UTX_v3.0 version (Sreedasyam et al. 2024) corrected the ~10 Mb inversion in chromosome D03 found between UTX v2.1 and other G. hirsutum genomes including ZM113.

Extended Data Fig. 6 Integrated analysis of 4 Mb inversion and centromere repositioning in D08 chromosome.

Shown left is the synteny plot of the chromosome D08 between ZM113 and other reference genomes. The inversion at 34–38 Mb divided the D08 locus into two haplotypes. GhSat194 arrays colocalize with the boundary of the inversion. Shown right are synteny plots between ZM113 with AD6 (classified to Hap1) and AD7 (classified to Hap2). CENH3 peaks were shown at the bottom.

Extended Data Fig. 7 IGV visualization of the ZM113 D08 chromosome showed log2(ChIP/input) signal tracks from CENH3 ChIP-seq experiments.

Top to bottom tracks are: 1-4. ZM113 ChIP-seq, GhCEN08 ___location, Ψ-GhD08CEN ___location, and repetitive sequence annotation; 5-7. ChIP-seq experiments on G. hirsutum (AD1) var TM-1 (ZJU), G. stephensii (AD7), G. ekmanianum (AD6), G. tomentosum (AD3), G. barbadense (AD2), G. darwinii (AD5), G. mustelinum (AD4), and G. raimondii (D5).

Extended Data Fig. 8 Comparative annotation and functional validation of the ZM113 private gene Ghir_D03G00738.

(a) IGV visualization of the TM-1 reference genome (version: CRI v1.0) region containing the unannotated Ghir_D03G00738 sequence. (b) IGV visualization of Ghir_D03G00738 annotation and transcriptome alignment in the ZM113 reference genome. (c) The VIGS results show that Ghir_D03G00738 regulates flowering time in upland cotton. Silenced plant phenotype images. (d) Silencing efficiency of Ghir_D03G00738 was detected by qRT-PCR. (e–g) Statistics of first fruiting node position, flowering time, and budding time.

Extended Data Fig. 9 PCR validation of the ZM113 private gene Ghir_D03G00738.

(a) Gene structure and PCR primer design. (b) Agarose gel image of the amplified fragments from both ZM113 and TM-1 materials. (c) Sequence alignment results of the amplified fragments. The unprocessed images of the gels are available in the source data file.

Source data

Extended Data Fig. 10 PCR amplification validation of D03_INV.

(a) Primer design scheme and sequences spanning the inversion breakpoint region. (b) Amplification results with Primer 1. (c) Amplification results with Primer 2. The unprocessed images of the gels are available in the source data file.

Source data

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Figs. 1–31.

Reporting Summary

Peer Review File

Supplementary Tables 1–30

Supplementary Table 1. Comparison of reference genomes of G. hirsutum. Supplementary Table 2. Multi-platform sequencing data generated for genome assembly. Supplementary Table 3. Summary of the five gap regions closed. Supplementary Table 4. Statistics of 26 chromosomes with centromere and telomere locations. Supplementary Table 5. Assembly consensus quality assessment by Mercury. Supplementary Table 6. Assembly and gene annotation quality assessment by BUSCO. Supplementary Table 7. Repetitive elements in ZM113 genome assembly. Supplementary Table 8. Statistics of functional annotation of gene models in the ZM113 genome assembly. Supplementary Table 9. Statistics of transcriptome support for gene models. Supplementary Table 10. Syntenic orthologous analysis of ZM113 genes relative to parental diploids. Supplementary Table 11. Annotation summary of non-coding RNA. Supplementary Table 12. Genomic distribution of rDNA arrays. Supplementary Table 13. Assembly features of ZM113 organelle genomes. Supplementary Table 14. Gene profile and organization of the ZM113 mtochondrial genome. Supplementary Table 15. Gene profile and organization of the ZM113 chloroplast genome. Supplementary Table 16. Summary of multi-omics data of ChIP-seq, BS-seq, and RNA-seq. Supplementary Table 17. Centromere positions in ZM113 identified by mapping centromere-specific repeat sequences. Supplementary Table 18. Gene identification within and flanking centromeric regions. Supplementary Table 19. Characterization of ZM113 centormeric genes by HOG, pan-genomic annotation, and differential expression. Supplementary Table 20. Statistics of repetitive sequences in centromeric regions and genome-wide. Supplementary Table 21. Characterization of 2530 CEN-Tes based on percentage content per centromere. Supplementary Table 22. DNA methylation levels, genome-wide and in centromeres. Supplementary Table 23. Summary of structurals variants between ZM113 and 9 other G. hirsutum genome assemblies used for genome comparisons. Supplementary Table 24. Summary of non-redundant SVs against ZM113. Supplementary Table 25. Summary of coreDELs relative to ZM113. Supplementary Table 26. Summary of 28 genes located in coreDELs. Supplementary Table 27. Candiate genes contraining SNPs and elite alleles associated with lint percentage (LP) and Flowering day (FD). Supplementary Table 28. SNPs and elite alleles associated with lint percentage (LP) and Flowering day (FD) in 419 accessions. Supplementary Table 29. The cumulative number of elite SNPs associated with LP and FD in 419 accessions. Supplementary Table 30. Haplotype typing results of the D03 chromosome interval.

Source data

Source Data Fig. 4

The unprocessed gels in red box correspond to Fig. 4g,h.

Source Data Extended Data Fig. 9

The unprocessed gels in red box correspond to Extended Data Fig. 9c.

Source Data Extended Data Fig. 10

The unprocessed gels in red box correspond to Extended Data Fig. 10b,c.

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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 57, 1031–1043 (2025). https://doi.org/10.1038/s41588-025-02130-4

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