In most studied eukaryotes, chromosomes are monocentric, with centromere activity confined to a single region. However, the rush family (Juncaceae) includes species with both monocentric (Juncus) and holocentric (Luzula) chromosomes, where centromere activity is distributed along the entire chromosome length. Here, we combine chromosome-scale genome assembly, epigenetic analysis, immuno-FISH and super-resolution microscopy to study the transition to holocentricity in Luzula sylvatica. We report repeat-based holocentromeres with an irregular distribution of features along the chromosomes. Luzula sylvatica holocentromeres are predominantly associated with two satellite DNA repeats (Lusy1 and Lusy2), while CENH3 also binds satellite-free gene-poor regions. Comparative repeat analysis suggests that Lusy1 plays a crucial role in centromere function across most Luzula species. Furthermore, synteny analysis between L. sylvatica (n = 6) and Juncus effusus (n = 21) suggests that holocentric chromosomes in Luzula could have arisen from chromosome fusions of ancestral monocentric chromosomes, accompanied by the expansion of CENH3-associated satellite repeats.

Biology Centre Czech Academy of Sciences Institute of Plant Molecular Biology České Budějovice Czech Republic

Cluster of Excellence on Plant Sciences Heinrich Heine University Düsseldorf Germany

Department of Chromosome Biology Max Planck Institute for Plant Breeding Research Cologne Germany

Department of Plant Developmental Genetics Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 61200 Brno Czech Republic

Laboratório de Citogenética e Evolução Vegetal Departamento de Botânica Centro de Biociências Universidade Federal de Pernambuco Recife PE 50670 901 Brazil

Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben 06466 Seeland Germany

Max Planck Genome Centre Max Planck Institute for Plant Breeding Research Cologne Germany

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Zobrazit více v PubMed

Talbert, P. B. & Henikoff, S. What makes a centromere? Exp. Cell Res.389, 111895 (2020). PubMed

Schubert, V. et al. Super-Resolution Microscopy Reveals Diversity of Plant Centromere Architecture. IJMS21, 3488 (2020). PubMed PMC

Heckmann, S. et al. The holocentric species Luzula elegans shows interplay between centromere and large‐scale genome organization. Plant J.73, 555–565 (2013). PubMed

Hofstatter, P. G. et al. Repeat-based holocentromeres influence genome architecture and karyotype evolution. Cell185, 3153–3168.e18 (2022). PubMed

Escudero, M., Marques, A., Lucek, K. & Hipp, A. L. Genomic hotspots of chromosome rearrangements explain conserved synteny despite high rates of chromosome evolution in a holocentric lineage. Mol. Ecol.10.1111/mec.17086 (2023). PubMed

Mata-Sucre, Y. et al. Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae). Front. Plant Sci.15, 1330927 (2024). PubMed PMC

Escudero, M., Márquez-Corro, J. I. & Hipp, A. L. The Phylogenetic Origins and Evolutionary History of Holocentric Chromosomes. Syst. Bot.41, 580–585 (2016).

Senaratne, A. P., Cortes-Silva, N. & Drinnenberg, I. A. Evolution of holocentric chromosomes: Drivers, diversity, and deterrents. Semin. Cell Dev. Biol.127, 90–99 (2022). PubMed

Drinnenberg, I. A., deYoung, D., Henikoff, S. & Malik, H. S. Recurrent loss of CenH3 is associated with independent transitions to holocentricity in insects. eLife3, e03676 (2014). PubMed PMC

Neumann, P. et al. Disruption of the standard kinetochore in holocentric Cuscuta species. Proc. Natl Acad. Sci. USA.120, e2300877120 (2023). PubMed PMC

Kuo, Y.-T., Schubert, V., Marques, A., Schubert, I. & Houben, A. Centromere diversity: How different repeat-based holocentromeres may have evolved. BioEssays46, e202400013 (2024). PubMed

Plohl, M., Meštrović, N. & Mravinac, B. Centromere identity from the DNA point of view. Chromosoma123, 313–325 (2014). PubMed PMC

Šatović-Vukšić, E. & Plohl, M. Satellite DNAs—From Localized to Highly Dispersed Genome Components. Genes14, 742 (2023). PubMed PMC

Hobza, R. et al. An accumulation of tandem DNA repeats on the Y chromosome in Silene latifolia during early stages of sex chromosome evolution. Chromosoma115, 376–382 (2006). PubMed

Kasinathan, S. & Henikoff, S. Non-B-Form DNA Is Enriched at Centromeres. Mol. Biol. Evol.35, 949–962 (2018). PubMed PMC

Marques, A. et al. Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin. Proc. Natl Acad. Sci. USA.112, 13633–13638 (2015). PubMed PMC

Kuo, Y.-T. et al. Holocentromeres can consist of merely a few megabase-sized satellite arrays. Nat. Commun.14, 3502 (2023). PubMed PMC

Ma, B. et al. The gap-free genome of mulberry elucidates the architecture and evolution of polycentric chromosomes. Horticult. Res.10, uhad111 (2023). PubMed PMC

Despot-Slade, E. et al. The Centromere Histone Is Conserved and Associated with Tandem Repeats Sharing a Conserved 19-bp Box in the Holocentromere of Meloidogyne Nematodes. Mol. Biol. Evol.38, 1943–1965 (2021). PubMed PMC

POWO. Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet; http://www.plantsoftheworldonline.org/ (2024).

Guerra, M., Ribeiro, T. & Felix, L. P. Monocentric chromosomes in Juncus ( Juncaceae) and implications for the chromosome evolution of the family. Bot. J. Linn. Soc.191, 475–483 (2019).

Mata-Sucre, Y. et al. Repeat-based phylogenomics shed light on unclear relationships in the monocentric genus Juncus L. ( Juncaceae). Mol. Phylogenet. cs Evol.189, 107930 (2023). PubMed

Dias, Y. et al. How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus ( Juncaceae). Plant J. tpj.16712 10.1111/tpj.16712 (2024). PubMed

Nagaki, K., Kashihara, K. & Murata, M. Visualization of Diffuse Centromeres with Centromere-Specific Histone H3 in the Holocentric Plant Luzula nivea. Plant Cell17, 1886–1893 (2005). PubMed PMC

Heckmann, S. et al. Holocentric Chromosomes of Luzula elegans Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region. Cytogenet Genome Res134, 220–228 (2011). PubMed

Bozek, M., Leitch, A. R., Leitch, I. J., Záveská Drábková, L. & Kuta, E. Chromosome and genome size variation in Luzula ( Juncaceae), a genus with holocentric chromosomes: Chromosome and C-Value Evolution in L uzula. Bot. J. Linn. Soc.170, 529–541 (2012).

Haizel, T., Lim, Y. K., Leitch, A. R. & Moore, G. Molecular analysis of holocentric centromeres of Luzula species. Cytogenet Genome Res109, 134–143 (2005). PubMed

Goodwin, Z. A. et al. The genome sequence of great wood-rush, Luzula sylvatica (Huds) Gaudin. Wellcome Open Res9, 124 (2024). PubMed PMC

Oliveira, L. et al. KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants. Chromosome Res32, 3 (2024). PubMed

Wang, J. et al. A high-quality chromosome-scale assembly of the centipedegrass [Eremochloa ophiuroides (Munro) Hack.] genome provides insights into chromosomal structural evolution and prostrate growth habit. Hortic. Res8, 201 (2021). PubMed PMC

Drábková, L. Z. A Survey of Karyological Phenomena in the Juncaceae with Emphasis on Chromosome Number Variation and Evolution. Bot. Rev.79, 401–446 (2013).

Lucek, K., Augustijnen, H. & Escudero, M. A holocentric twist to chromosomal speciation? Trends Ecol. Evol.37, 655–662 (2022). PubMed

Cortes-Silva, N. et al. CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T. Curr. Biol.30, 561–572.e10 (2020). PubMed

Jankowska, M. et al. Holokinetic centromeres and efficient telomere healing enable rapid karyotype evolution. Chromosoma124, 519–528 (2015). PubMed

Castellani, M. et al. Meiotic recombination dynamics in plants with repeat-based holocentromeres shed light on the primary drivers of crossover patterning. Nat. Plants10, 423–438 (2024). PubMed PMC

Souza, T. B. D. et al. Distinct patterns of satDNA distribution in holocentric chromosomes of spike-sedges (Eleocharis, Cyperaceae). Genome gen-2024-0089 10.1139/gen-2024-0089 (2024). PubMed

Liu, H. et al. The genome of Eleocharis vivipara elucidates the genetics of C 3 –C 4 photosynthetic plasticity and karyotype evolution in the Cyperaceae. JIPB jipb.13765 10.1111/jipb.13765 (2024). PubMed

Ribeiro, T. et al. Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species. Chromosoma126, 325–335 (2016). PubMed

Costa, L., Marques, A., Buddenhagen, C. E., Pedrosa-Harand, A. & Souza, G. Investigating the diversification of holocentromeric satellite DNA Tyba in Rhynchospora (Cyperaceae). Ann. Bot.131, 813–825 (2023). PubMed PMC

Wlodzimierz, P. et al. Cycles of satellite and transposon evolution in Arabidopsis centromeres. Nature618, 557–565 (2023). PubMed

Hiatt, E. N., Kentner, E. K. & Dawe, R. K. Independently Regulated Neocentromere Activity of Two Classes of Tandem Repeat Arrays. Plant Cell14, 407–420 (2002). PubMed PMC

Piras, F. M. et al. Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus. PLoS Genet6, e1000845 (2010). PubMed PMC

Cappelletti, E. et al. Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras. Mol. Biol. Evol.39, msac162 (2022). PubMed PMC

Ning, Y. et al. The chromosome-scale genome of Kobresia myosuroides sheds light on karyotype evolution and recent diversification of a dominant herb group on the Qinghai-Tibet Plateau. DNA Res.30, dsac049 (2023). PubMed PMC

Wright, C. J., Stevens, L., Mackintosh, A., Lawniczak, M. & Blaxter, M. Comparative genomics reveals the dynamics of chromosome evolution in Lepidoptera. Nat. Ecol. Evol.10.1038/s41559-024-02329-4 (2024). PubMed PMC

Augustijnen, H. et al. A macroevolutionary role for chromosomal fusion and fission in Erebia butterflies. Sci. Adv.10, eadl0989 (2024). PubMed PMC

Elliott, T. L. & Davies, T. J. Phylogenetic attributes, conservation status and geographical origin of species gained and lost over 50 years in a UNESCO Biosphere Reserve. Biodivers. Conserv28, 711–728 (2019).

Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods18, 170–175 (2021). PubMed PMC

Seppey, M., Manni, M. & Zdobnov, E. M. BUSCO: Assessing Genome Assembly and Annotation Completeness. in Gene Prediction (ed. Kollmar, M.) 1962 227–245 (Springer New York, New York, NY, 2019). PubMed

Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: quality assessment tool for genome assemblies. Bioinformatics29, 1072–1075 (2013). PubMed PMC

Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics25, 1754–1760 (2009). PubMed PMC

Ghurye, J. et al. Integrating Hi-C links with assembly graphs for chromosome-scale assembly. PLoS Comput Biol.15, e1007273 (2019). PubMed PMC

Durand, N. C. et al. Juicebox Provides a Visualization System for Hi-C Contact Maps with Unlimited Zoom. Cell Syst.3, 99–101 (2016). PubMed PMC

Dudchenko, O. et al. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science356, 92–95 (2017). PubMed PMC

Sun, H., Ding, J., Piednoël, M. & Schneeberger, K. findGSE: estimating genome size variation within human and Arabidopsis using k -mer frequencies. Bioinformatics34, 550–557 (2018). PubMed

Marçais, G. & Kingsford, C. A fast, lock-free approach for efficient parallel counting of occurrences of k -mers. Bioinformatics27, 764–770 (2011). PubMed PMC

Ma, W. et al. The distribution of α-kleisin during meiosis in the holocentromeric plant Luzula elegans. Chromosome Res24, 393–405 (2016). PubMed

Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j.17, 10 (2011).

Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods9, 357–359 (2012). PubMed PMC

Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res44, W160–W165 (2016). PubMed PMC

Lopez-Delisle, L. et al. pyGenomeTracks: reproducible plots for multivariate genomic datasets. Bioinformatics37, 422–423 (2021). PubMed PMC

Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biol.9, R137 (2008). PubMed PMC

Stovner, E. B. & Sætrom, P. epic2 efficiently finds diffuse domains in ChIP-seq data. Bioinformatics35, 4392–4393 (2019). PubMed

Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics27, 1571–1572 (2011). PubMed PMC

Novák, P., Neumann, P. & Macas, J. Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2. Nat. Protoc.15, 3745–3776 (2020). PubMed

Novák, P., Hoštáková, N., Neumann, P. & Macas, J. DANTE and DANTE_LTR: lineage-centric annotation pipelines for long terminal repeat retrotransposons in plant genomes. NAR Genom. Bioinforma.6, lqae113 (2024). PubMed PMC

Neumann, P. et al. Plant centromeric retrotransposons: a structural and cytogenetic perspective. Mob. DNA2, 4 (2011). PubMed PMC

Neumann, P., Novák, P., Hoštáková, N. & Macas, J. Systematic survey of plant LTR-retrotransposons elucidates phylogenetic relationships of their polyprotein domains and provides a reference for element classification. Mob. DNA10, 1 (2019). PubMed PMC

Novák, P. et al. TAREAN: a computational tool for identification and characterization of satellite DNA from unassembled short reads. Nucleic Acids Res.45, e111 (2017). PubMed PMC

Sonnhammer, E. L. L. & Durbin, R. A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene167, GC1–GC10 (1995). PubMed

Kearse, M. et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics28, 1647–1649 (2012). PubMed PMC

Yu, Y., Ouyang, Y. & Yao, W. shinyCircos: an R/Shiny application for interactive creation of Circos plot. Bioinformatics34, 1229–1231 (2018). PubMed

Sweeten, A. P., Schatz, M. C. & Phillippy, A. M. ModDotPlot—rapid and interactive visualization of tandem repeats. Bioinformatics40, btae493 (2024). PubMed PMC

Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods17, 261–272 (2020). PubMed PMC

Hunter, J. D. Matplotlib: A 2D Graphics Environment. Comput. Sci. Eng.9, 90–95 (2007).

McKinney, W. pandas: a foundational Python library for data analysis and statistics. Python high. Perform. Sci. Comput.14, 1–9 (2011).

Dale, R. K., Pedersen, B. S. & Quinlan, A. R. Pybedtools: a flexible Python library for manipulating genomic datasets and annotations. Bioinformatics27, 3423–3424 (2011). PubMed PMC

Harris, C. R. et al. Array programming with NumPy. Nature585, 357–362 (2020). PubMed PMC

Waskom, M. seaborn: statistical data visualization. JOSS6, 3021 (2021).

Lyons, E., Pedersen, B., Kane, J. & Freeling, M. The Value of Nonmodel Genomes and an Example Using SynMap Within CoGe to Dissect the Hexaploidy that Predates the Rosids. Trop. Plant Biol.1, 181–190 (2008).

Soderlund, C., Bomhoff, M. & Nelson, W. M. SyMAP v3.4: a turnkey synteny system with application to plant genomes. Nucleic Acids Res.39, e68 (2011). PubMed PMC

Lovell, J. T. et al. GENESPACE tracks regions of interest and gene copy number variation across multiple genomes. eLife11, e78526 (2022). PubMed PMC

Hao, Z. et al. RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput. Sci.6, e251 (2020). PubMed PMC

Kanduri, C., Bock, C., Gundersen, S., Hovig, E. & Sandve, G. K. Colocalization analyses of genomic elements: approaches, recommendations and challenges. Bioinformatics35, 1615–1624 (2019). PubMed PMC

Weisshart, K., Fuchs, J. & Schubert, V. Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei. BIO-PROTOCOL6, (2016).

Krátká, M. Repeat-based-holocentromeres-of-Luzula-sylvatica. Zenodo10.5281/ZENODO.13945236 (2024). PubMed PMC

Repeat-based holocentromeres of the woodrush Luzula sylvatica reveal insights into the evolutionary transition to holocentricity