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The complete genome assembly of Nicotiana benthamiana reveals the genetic and epigenetic landscape of centromeres

Nature Plants volume 10, pages 1928–1943 (2024) Cite this article

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Abstract

Nicotiana benthamiana is a model organism widely adopted in plant biology. Its complete assembly remains unavailable despite several recent improvements. To further improve its usefulness, we generate and phase the complete 2.85 Gb genome assembly of allotetraploid N. benthamiana. We find that although Solanaceae centromeres are widely dominated by Ty3/Gypsy retrotransposons, satellite-based centromeres are surprisingly common in N. benthamiana, with 11 of 19 centromeres featured by megabase-scale satellite arrays. Interestingly, the satellite-enriched and satellite-free centromeres are extensively invaded by distinct Gypsy retrotransposons which CENH3 protein more preferentially occupies, suggestive of their crucial roles in centromere function. We demonstrate that ribosomal DNA is a major origin of centromeric satellites, and mitochondrial DNA could be employed as a core component of the centromere. Subgenome analysis indicates that the emergence of satellite arrays probably drives new centromere formation. Altogether, we propose that N. benthamiana centromeres evolved via neocentromere formation, satellite expansion, retrotransposon enrichment and mtDNA integration.

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Fig. 1: Whole-genome landscape of the N. benthamiana complete genome.
The alternative text for this image may have been generated using AI.
Fig. 2: Subgenome assignments of the allotetraploid N. benthamiana and tracing the potential diploid ancestors.
The alternative text for this image may have been generated using AI.
Fig. 3: Genetic and epigenetic landscape of centromeres in N. benthamiana.
The alternative text for this image may have been generated using AI.
Fig. 4: Satellites drive the formation of neocentromere in N. benthamiana.
The alternative text for this image may have been generated using AI.
Fig. 5: Precise kinetochore assembly sites and epigenetic centromere landscapes.
The alternative text for this image may have been generated using AI.
Fig. 6: Scenario for the palaeohistory of N. benthamiana genome evolution.
The alternative text for this image may have been generated using AI.

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Data availability

The raw sequencing data and the genome assembly have been deposited at the China National Center for Bioinformation (https://ngdc.cncb.ac.cn/) under project number PRJCA022857. The nuclear genome assembly and annotation are also available in Zenodo93. The mitochondria and chloroplast genomes have been deposited at the China National Center for Bioinformation under accession number C_AA066595 and C_AA066594, respectively.

Code availability

This manuscript does not report original code.

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Acknowledgements

We thank the Bioinformatics Platform at Peking University Institute of Advanced Agricultural Sciences for providing the high-performance computing resources. This work was supported by the Key R&D Program of Shandong Province (ZR202211070163 to L.G.), the Taishan Scholars Program and Natural Science Foundation for Distinguished Young Scholars (ZR2023JQ010 to L.G.) of Shandong Province.

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Authors and Affiliations

  1. Shandong Key Laboratory of Precision Molecular Crop Design and Breeding, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences in Weifang, Weifang, China

    Weikai Chen, Ming Yan, Shaoying Chen, Jie Sun, Jingxuan Wang, Dian Meng, Jun Li, Lili Zhang & Li Guo

  2. College of Modern Agriculture and Environment, Weifang Institute of Technology, Weifang, China

    Lili Zhang

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Contributions

L.G. conceived and supervised the project. W.C. performed genome assembly and analysis of centromeric sequences. M.Y. prepared figures and tables. S.C., J.S. and J.W. performed epigenetic analysis. D.M. conducted epigenome sequencing. J.L. and L.Z. generated genome sequencing data. W.C. and L.G. wrote the paper. All authors read and approved the final version of the paper.

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Correspondence to Li Guo.

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Extended data

Extended Data Fig. 1 Whole-genome coverage of HiFi reads across the NB.T2T assembly.

Local coverage-anomalous regions were shown in black lines. The regions of rDNA arrays, satellites, NUPTs and NUMTs were marked on the bottom track. The red triangles and black asterisks represent the gaps in HiFi assembly and HiFi & ONT assembly before gap-closure, respectively.

Extended Data Fig. 2 Genome structure of the telomeres and subtelomeres.

a, A presentation of the subtelomere alignment between two assemblies. The collinear regions were linked by gray lines. The assembly gaps in NB.PCP were marked by black lines. The following information exhibited the HiFi coverage, ONT coverage, 5mC level, gene number and TE distribution calculated in 20-kb bins. The heatmaps showed the pairwise sequence identity (%) between 5-kb bins of the subtelomeric regions in Chr01. The histogram showed the satellite repeat length across all subtelomeric regions. Tsate181 and Tsate164 represent 181-bp and 164-bp subtelomeric satellite, respectively. b, Length of telomeres and subtelomeres in 19 chromosomes. c, Maximum-likelihood phylogenetic tree of Tsate181 sampled from 16 subtelomeres (L, left terminal; R, right terminal) and three interstitial subtelomeric sequences (marked as M), rooted using Tsate164. The color represents the origin of each satellite in chromosome location.

Extended Data Fig. 3 Phased subgenomes of allotetraploid N. benthamiana.

a, Alignments of N. benthamiana chromosomes with itself. b, Circos plot of subgenome partitions of N. benthamiana. Track from outer to inner: subgenome assignments by a k-means algorithm, significant enrichment of subgenome specific k-mers, normalized proportion of subgenome specific k-mers, count of B subgenome specific k-mers, count of A subgenome specific k-mers, count of subgenome specific LTR-RTs (in yellow and blue color), and homoeologous blocks of each homoeologous chromosome set. c-d, Alignments of N. benthamiana chromosomes with its potential diploid ancestors of N. sylvestris (c) and N. attenuata (d).

Extended Data Fig. 4 CENH3 protein alignments and CENH3 ChIP-seq values across whole genome.

a, Two antibodies against N-terminal (ARTKHLALRKQSRPPSRPTA) or whole protein of CENH3 were synthesized and used to conduct ChIP-seq experiment. b, CENH3 log2(ChIP/control) enrichment level across 19 chromosomes. The diamond represents the position of identified centromeres.

Extended Data Fig. 5 CENH3 enrichment, repeat distribution and StainedGlass heatmap of the 19 centromeres.

The CENH3 panels show the log2(ChIP/control ratio) calculated in 20-kb bins in 4 Mb or 9 Mb windows for centromere lower than or higher than 3 Mb, respectively. The heatmaps show the pairwise sequence identity (%) between 5-kb sequences.

Extended Data Fig. 6 Genetic and epigenetic landscape of NUMTs-type and satellite-type centromeres.

a-b, Schematic representation showing the CENH3 enrichment level, GC content, and distribution of different repeats in 20-kb bins across Chr02: 15.00-17.58 Mb (a) and Chr08: 118.74-123.12 Mb (b). The bottom showing a conserved CEN02-spaning synteny block, and a StainedGlass sequence identity heatmap of CEN08.

Extended Data Fig. 7 Identification of centromeric satellite repeats and phylogenetic analysis of Gypsy retrotransposons.

a, Circos plot showed the distribution of different satellite family. b, Pairwise sequence alignments of 19 centromeres. The red and blue lines indicate forward- and reverse-strand similarity, respectively. c, Maximum likelihood phylogenetic tree of intact Gypsy retrotransposons, colored according to seven subfamilies. Asterisks at the branch indicate elements within the centromeres.

Extended Data Fig. 8 Satellites drive the formation of neocentromere (CEN10) in N. benthamiana.

a, A synteny block was conserved among N. benthamiana, tobacco, potato and pepper, which spanned the centromere CEN10 of N. benthamiana. b, CENH3 enrichment and Gypsy density in 20 kb windows within CEN10-spanning synteny block between Chr09 and Chr10. Full descriptions for the panels are also available in the main Fig. 4 legend. StainedGlass sequence identity heatmaps were shown beneath.

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Supplementary Tables 1–13.

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Chen, W., Yan, M., Chen, S. et al. The complete genome assembly of Nicotiana benthamiana reveals the genetic and epigenetic landscape of centromeres. Nat. Plants 10, 1928–1943 (2024). https://doi.org/10.1038/s41477-024-01849-y

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