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Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing

Nature Genetics volume 57, pages 2028–2039 (2025)Cite this article

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Abstract

Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.

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Fig. 1: A comprehensive overview of the genetic transformation system of cotton and the complete genome features of Jin668 and YZ1.
Fig. 2: Completeness and accuracy validation of both chromosome arms and centromeres in Jin668 genome.
Fig. 3: Centromeric characteristics of Jin668.
Fig. 4: In-depth comparative analysis of the Jin668 versus TM-1, YZ1 and ZM24.
Fig. 5: Overview of chromatin accessibility and transcriptome dynamics in Jin668 and TM-1 during SE.
Fig. 6: Functional verification of regeneration-related genes.
Fig. 7: Cotton genetic variation substantially impacts the efficacy of CRISPR-based genome editing.

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Data availability

The T2T-Jin668 and YZ1 genome assemblies and annotation data are available at NCBI (PRJNA874817 and PRJNA960814) and T2TCotton-Hub (http://jinlab.hzau.edu.cn/T2TCottonHub/ or http://cotton.hzau.edu.cn/T2TCottonHub/). The raw sequencing data used for de novo whole-genome assembly of Jin668 and YZ1 are available in NCBI under BioProjects PRJNA874817 and PRJNA960814, respectively. RNA-seq of Jin668 and YZ1 are available in NCBI under BioProjects PRJNA874819 and PRJNA960820, respectively. ATAC–seq data of Jin668 is available in NCBI under BioProjects PRJNA960832. ATAC–seq and RNA-seq data for TM-1 during SE are available in NCBI under BioProjects PRJNA960828 and PRJNA960825, respectively. ATAC–seq and RNA-seq data for YZ1 during SE are available in NCBI under BioProjects PRJNA1059614 and PRJNA1059613, respectively. ATAC–seq and RNA-seq data for ZB1092 during SE are available in NCBI under BioProjects PRJNA1059611 and PRJNA1059609, respectively. The ChIP–seq data for Jin668 and YZ1 are uploaded to BioProjects PRJNA1079680 and PRJNA1079682, respectively. Seeds of Jin668 and YZ1 used in this study can be obtained from the corresponding author upon request. The reference genome assembly and annotation files of TM-1 (v3.1) used in this study were downloaded from https://phytozome-next.jgi.doe.gov/ and are also accessible from T2TCottonHub. Additionally, all available SE-related motifs were obtained from Plant Transcription Factor Database (v5.0; https://planttfdb.gao-lab.org/). Further details on data accessibility are outlined in the Supplementary Methods and Methods. Source data are provided with this paper.

Code availability

All original codes used in this article are available via Zenodo at https://doi.org/10.5281/zenodo.15035095 (ref. 96) and GitHub (https://github.com/tiramisutes/T2T-Cotton-Genomes).

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Acknowledgements

The study was supported by grants from the National Science Fund for Distinguished Young Scholars (32325039 to S.J.) and the Young Scientists Fund under the National Natural Science Foundation of China (32201856 to Z.X.). The computations in this paper were run on the bioinformatics computing platform of the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

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Author notes

  1. These authors contributed equally: Zhongping Xu, Guanying Wang.

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Zhongping Xu, Guanying Wang, Xiangqian Zhu, Ruipeng Wang, Longfu Zhu, Lili Tu, Maojun Wang, Xianlong Zhang & Shuangxia Jin

  2. Research Base, Anyang Institute of Technology, State Key Laboratory of Cotton Biology, Anyang, China

    Yuling Liu & Renhai Peng

  3. Department of Biosciences, Durham University, Durham, UK

    Keith Lindsey

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  1. Zhongping Xu
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Contributions

S.J. and X. Zhang designed and supervised the research. Z.X. and G.W. collected materials for genome and transcriptome sequencing. Z.X. and G.W. collected materials for ATAC–seq. Z.X. and R.W. performed bioinformatics analysis. Y.L. and R.P. performed the FISH experiment. G.W. and X.Z. performed the gene editing experiments. M.W., L.T. and L.Z. contributed to the project discussion. Z.X. and G.W. wrote the manuscript with input from all other authors. S.J., K.L., X. Zhang and M.W. edited the manuscript. All authors have read and approved the manuscript.

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Correspondence to Maojun Wang, Xianlong Zhang or Shuangxia Jin.

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Nature Genetics thanks Amanda Hulse-Kemp, Jinsheng Lai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Genomic characterization of Jin668 and YZ1.

The k-mer (31-mer) frequency distribution was estimated by GenomeScope2.0 analysis of Jin668 (a) and YZ1 (b) genome. c, The distribution of telomeres, centromeres, and gaps regions in the genomes of Jin668 and YZ1.

Source data

Extended Data Fig. 2 Completeness and accuracy validation of chromosome arms and centromeres in the Jin668 genome.

a, Assessment of the Jin668 genome assembly using LTR Assembly Index (LAI). The x-axis lists chromosomes and y-axis represents LAI values. The red dashed line represents the mean value. b,c, Hi-C map of the Jin668 genome showing genome- and chromosome-wide all-by-all interactions. d, Bionano de novo assembly contigs were mapped to the Jin668 reference assembly. e, The distribution of QV values calculated from Merqury and Merfin on different chromosomes. f, The distribution of different types of unique k-mers along the centromeric regions in chromosomes of Jin668. Each bar shows the number of different types of k-mers in a bin of length 20 Kb. The blue bars represent single-clump k-mers, which suggest a good base-level quality. While the orange (multiple-clumps) and green (no-clumps) bars suggest a low base-level quality in the region. The x-axis unit is megabases (Mb), and the y-axis represents values multiplied by 10³. g, NucFreq plot of centromeric regions in chromosomes of Jin668. HiFi coverage depth (black) along with secondary allele frequency (red) for all centromeres and surrounding regions. The x-axis represents chromosome positions in megabases (Mb), and the y-axis shows HiFi depth, ranging from 0 to 100.

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Extended Data Fig. 3 Segmental duplication (SD) content of the Jin668 genome.

a,b, The intrachromosome SDs with a length greater or less than 5 kb. c, Comparison of SD length and identity in different regions of the genome. The identity (top) and length (bottom) of SDs across commonly delineated regions of the genome (colors).

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Extended Data Fig. 4 The percentage of AT/GC base composition, TEs, 5mC DNA methylation, histone modification and 3D genome architecture within the centromeres.

Quantification of genomic features plotted along chromosome arms that were proportionally scaled between telomeres (TEL) and centromere midpoints (CEN).

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Extended Data Fig. 5 A comparative genome analysis of the Jin668 versus TM-1, YZ1, and ZM24.

a, Genome-wide syntenic relationships among At and Dt subgenomes in four Upland cotton accessions relative to the A-genome-like Ga (A2 genome) and D-genome-like Gr (D5 genome). b, The distribution of variation density between Jin668 versus TM-1, YZ1, and ZM24 genomes. c, The distribution of SE-related genes and gene density, as well as genomic variations from Fig.1, on the chromosomes of the Jin668 genome. The circos plot from outside to inside shows: chromosome, SE-related genes, gene density in Jin668, SNPs, InDels, PAVs, inversions and translocation density between Jin668 with TM-1. d, The correlation between SNPs, InDels, PAVs, inversions and translocations with SE-related genes. The significance was tested using the overlapPermTest function in regioneR (https://www.bioconductor.org/packages/devel/bioc/html/regioneR.html), which calls permTest with the appropriate parameters to perform the permutation test. SE, somatic embryogenesis.

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Extended Data Fig. 6 Sample preparation, sequencing, and quality evaluation of ATAC library.

a, Schematic outline of genome-wide ATAC–seq and RNA-seq assays and time points of sample collection with two independent biological replicates, as well as the roadmap of an integration analysis. HAI, hours after inoculation. b, The principal component plots of ATAC–seq data sequenced from Jin668, YZ1, TM-1 and ZB1092, respectively. Color code is shown. Each dot represents one sample. c, The genome-wide distribution of ATAC–seq peaks for Jin668, YZ1, TM-1 and ZB1092. Window size: TSS ± 3.0 Kb. d, The distribution of SPOT values for all samples from Jin668, YZ1, TM-1 and ZB1092, respectively. e, The distribution of FRiP values for all samples from Jin668, YZ1, TM-1 and ZB1092, respectively. f, ATAC–seq profiling in Jin668, YZ1, TM-1 and ZB1092. Left, the number statistics of chromatin accessibility peaks on each chromosome. Middle, the bar chart shows the total number of peaks detected at the corresponding induction time point. Right, the pie chart illustrates the percentage distribution of peaks across various genomic regions.

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Extended Data Fig. 7 Identification of SE-related genes.

Identification of SE-related genes based on genome information, chromatin accessibility and gene expression change trend at different time points of SE stage, and other external data (published literature and data generated in our laboratory earlier, etc.).

Extended Data Fig. 8 AGL promotes cotton regeneration.

a, Transcription and epigenetic tracks for AGL. Transcription data shown as mean ± s.d. of 3 biological replicates. b, The CPR of CRISPR edited, overexpression and control lines at 60 days post-induction. Data are represented as mean ± s.d. Differences between groups were evaluated by two-sided Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 independent biological replicates. The P7N was used as control. c, The paraffin sections of CRISPR edited, overexpression and control lines for Jin668 and TM-1 at different days post-induction. Scale bars represent 100 μm.

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Extended Data Fig. 9 Genetic transformation experiments demonstrate that the Jin668 genome enhances the accuracy of sgRNA design for CRISPR gene editing.

a, The sequence homology of a putative sgRNA (present in Jin668 but absent in TM-1) between Jin668 and TM-1. b,c, The mutational landscape of the genomic regions corresponding to targets sgRNA within the callus of Jin668 (b) and TM-1 (c) after edited using CRISPR/Cas9. The percentage signifies the ratio of reads corresponding to this mutation type to the total read count.

Extended Data Fig. 10 Gene expression dynamics across 15 tissues in Jin668 and YZ1.

a, The profile of genes expressed in all 15 tissues of Jin668 (left) and YZ1 (right). b, Statistics on the number of genes co-expressed in multiple tissues of Jin668 (left) and YZ1 (right). c, Statistics on the number of genes specifically expressed in each tissue of Jin668 (left) and YZ1 (right). Each tissue is represented by the number corresponding to that shown in the graphic illustration in a.

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Supplementary information

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Supplementary Figs. 1–31, Supplementary Notes 1–13 and Supplementary Methods.

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Supplementary Tables 1–35.

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Xu, Z., Wang, G., Zhu, X. et al. Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing. Nat Genet 57, 2028–2039 (2025). https://doi.org/10.1038/s41588-025-02258-3

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