Horticulture Featured
| Open Access | A Conserved Centromeric DNA Signature Underpins Profound Karyotype Stability in Diverse Citrus Species
Dr. Elena Petrova , Department of Plant and Microbial Biology, Wageningen University & Research, Wageningen, Netherlands Dr. Kenji Tanaka , Center for Plant Science Innovation, Nara Institute of Science and Technology, Nara, Japan Prof. Samuel Jones , Citrus Research and Education Center, Department of Horticultural Sciences, University of Florida, Gainesville, USAAbstract
Background: The Citrus genus, renowned for its economic importance, possesses a complex evolutionary history shaped by widespread interspecific hybridization. A detailed understanding of its genome structure is essential for both evolutionary studies and crop improvement. However, high-resolution, comparative karyotype analyses across the genus are lacking, and the DNA sequences that define functional Citrus centromeres—critical for genomic stability—remain poorly understood.
Methods: We developed a set of chromosome-specific oligonucleotide (oligo) probes based on the sweet orange (Citrus sinensis) reference genome. These probes were used to perform multicolor oligo-fluorescence in situ hybridization (oligo-FISH), or chromosome painting, on mitotic chromosomes from several ancestral and cultivated Citrus species. Concurrently, we employed chromatin immunoprecipitation with an antibody against the centromeric histone CENH3, followed by sequencing (ChIP-seq), to identify the core DNA sequences of functional centromeres.
Results: The oligo-probes successfully painted and distinguished all nine chromosome pairs in the analyzed species. Comparative karyotyping revealed a remarkable degree of conservation in chromosome number (2n=18), size, and morphology across the genus, indicating profound karyotypic stasis despite extensive hybridization. Our ChIP-seq analysis identified a highly conserved 165-bp satellite repeat (CitCEN165) as the dominant CENH3-associated sequence. Subsequent FISH experiments using a CitCEN165 probe confirmed its exclusive localization to the primary constriction of every chromosome in all tested species.
Conclusion: Our findings demonstrate that Citrus evolution has proceeded with minimal large-scale chromosomal rearrangement, a feature that likely facilitated its reticulate evolution. The identification and validation of the conserved CitCEN165 repeat as the primary centromeric DNA element provides a fundamental insight into Citrus genome architecture. This study offers a powerful cytogenomic toolkit and foundational knowledge that will significantly benefit future genetic research and advanced breeding strategies in Citrus.
Keywords
Citrus, Chromosome Painting, Oligo-FISH, Karyotype Evolution
References
Albert PS, Zhang T, Semrau K, Rouillard JM, Kao YH, Wang CJR, et al. Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships. Proc Natl Acad Sci USA. 2019;116:1679–85. https://doi.org/10.1073/pnas.1813957116.
Braz GT, He L, Zhao H, Zhang T, Semrau K, Rouillard JM, et al. Comparative oligo-FISH mapping: an efficient and powerful methodology to reveal karyotypic and chromosomal evolution. Genetics. 2018;208:513–23. https://doi.org/10.1534/genetics.117.300344.
Braz GT, Do Vale Martins L, Zhang T, Albert PS, Birchler JA, Jiang J. A universal chromosome identification system for maize and wild zea species. Chromosome Res. 2020;28:183–94. https://doi.org/10.1007/s10577-020-09630-5.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. https://doi.org/10.1186/1471-2105-10-421.
[5] Carvalho R, Soares Filho WS, Brasileiro-Vidal AC, Guerra M. The relationships among lemons, limes and citron: a chromosomal comparison. Cytogenet Genome Res. 2005;109:276–82. https://doi.org/10.1159/000082410.
Cheng Z, Dong F, Langdon T, Ouyang S, Buell CR, Gu M, et al. Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. Plant Cell. 2002;14:1691–704. https://doi.org/10.1105/tpc.003079.
Comai L, Maheshwari S, Marimuthu MPA. Plant centromeres. Curr Opin Plant Biol. 2017;36:158–67. https://doi.org/10.1016/j.pbi.2017.03.003.
Deng H, Tang G, Xu N, Gao Z, Lin L, Liang D, et al. Integrated karyotypes of diploid and tetraploid carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) as determined by sequential multicolor fluorescence in situ hybridization 1with tandemly repeated DNA sequences. Front Plant Sci. 2020;11:569. https://doi.org/10.3389/fpls.2020.00569.
Do Vale Martins L, Yu F, Zhao H, Dennison T, Lauter N, Wang H, et al. Meiotic crossovers characterized by haplotype-specific chromosome painting in maize. Nat Commun. 2019;10:4604. https://doi.org/10.1038/s41467-019-12646-z.
Gore MA, Chia JM, Elshire RJ, Sun Q, Ersoz ES, Hurwitz BL, et al. A first-generation haplotype map of maize. Science. 2009;326:1115–7. https://doi.org/10.1126/science.1177837.
Guerra M. Cytogenetics of rutaceae. V. High chromosomal variability in Citrus species revealed by CMA/DAPI staining. Heredity. 1993;71:234–41. https://doi.org/10.1038/hdy.1993.131.
Han Y, Zhang T, Thammapichai P, Weng Y, Jiang J. Chromosome-specific painting in cucumis species using bulked oligonucleotides. Genetics. 2015;200:771–9. https://doi.org/10.1534/genetics.115.177642.
Hao Z, Lv D, Ge Y, Shi J, Weijers D, Yu G, et al. RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput Sci. 2020;6: e251. https://doi.org/10.7717/peerj-cs.251.
Harun A, Liu H, Song S, Asghar S, Wen X, Fang Z, et al. Oligonucleotide fluorescence in situ hybridization: an efficient chromosome painting method in plants. Plants (Basel). 2023;12:2816. https://doi.org/10.3390/plants12152816.
He L, Zhao H, He J, Yang Z, Guan B, Chen K, et al. Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome-specific painting. Plant J. 2020;103:2225–35. https://doi.org/10.1111/tpj.14894.
Jiang J. Fluorescence in situ hybridization in plants: recent developments and future applications. Chromosome Res. 2019;27:153–65. https://doi.org/10.1007/s10577-019-09607-z.
Kato A, Lamb JC, Birchler JA. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc Natl Acad Sci USA. 2004;101:13554–9. https://doi.org/10.1073/pnas.0403659101.
Kim JS, Childs KL, Islam-Faridi MN, Menz MA, Klein RR, Klein PE, et al. Integrated karyotyping of sorghum by in situ hybridization of landed BACs. Genome. 2002;45:402–12. https://doi.org/10.1139/g01-141.
Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. https://doi.org/10.1038/nmeth.1923.
Levan A, Fredga K, Sandberg AA. Nomenclature for centromeric position on chromosomes. Hereditas. 2009;52:201–20. https://doi.org/10.1111/j.1601-5223.1964.tb01953.x.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9. https://doi.org/10.1093/bioinformatics/btp352.
Ma H, Ding W, Chen Y, Zhou J, Chen W, Lan C, et al. Centromere plasticity with evolutionary conservation and divergence uncovered by wheat 10+ genomes. Mol Biol Evol. 2023;40:msad176. https://doi.org/10.1093/molbev/msad176.
Melters DP, Bradnam KR, Young HA, Telis N, May MR, Ruby JG, et al. Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution. Genome Biol. 2013;14:R10. https://doi.org/10.1186/gb-2013-14-1-r10.
Nagaki K, Talbert PB, Zhong C, Dawe RK, Henikoff S, Jiang J. Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of arabidopsis thaliana centromeres. Genetics. 2003;163:1221–5. https://doi.org/10.1093/genetics/163.3.1221.
Neph S, Kuehn MS, Reynolds AP, Haugen E, Thurman RE, Johnson AK, et al. BEDOPS: high-performance genomic feature operations. Bioinformatics. 2012;28:1919–20. https://doi.org/10.1093/bioinformatics/bts277.
Oliveira LC, Torres GA. Plant centromeres: genetics, epigenetics and evolution. Mol Biol Rep. 2018;45:1491–7. https://doi.org/10.1007/s11033-018-4284-7.
Plohl M, Meštrović N, Mravinac B. Centromere identity from the DNA point of view. Chromosoma. 2014;123:313–25. https://doi.org/10.1007/s00412-014-0462-0.
Song S, Liu H, Miao L, He L, Xie W, Lan H, et al. Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis. J Genet Genomics. 2023a;50:410–21. https://doi.org/10.1016/j.jgg.2022.12.003.
Song S, Liu H, Miao L, Lan H, Chen C. Centromeric repeats in Citrus sinensis provide new insights into centromeric evolution and the distribution of G-quadruplex structures. Hortic Adv. 2023b;1:7. https://doi.org/10.1007/s44281-023-00010-7.
Su H, Liu Y, Liu C, Shi Q, Huang Y, Han F. Centromere satellite repeats have undergone rapid changes in polyploid wheat subgenomes. Plant Cell. 2019;31:2035–51. https://doi.org/10.1105/tpc.19.00133.
Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14:178–92. https://doi.org/10.1093/bib/bbs017.
Wang L, Huang Y, Liu Z, He J, Jiang X, He F, et al. Somatic variations led to the selection of acidic and acidless orange cultivars. Nat Plants. 2021;7:954–65. https://doi.org/10.1038/s41477-021-00941-x.
Wang Y, Zhou F, Li Y, Yu X, Wang Y, Zhao Q, et al. Characterization of the CsCENH3 protein and centromeric DNA profiles reveal the structures of centromeres in cucumber. Hortic Res. 2024;11:uhae127. https://doi.org/10.1093/hr/uhae127.
Wu GA, Terol J, Ibanez V, López-García A, Pérez-Román E, Borredá C, et al. Genomics of the origin and evolution of Citrus. Nature. 2018;554:311–6. https://doi.org/10.1038/nature25447.
Xia QM, Miao LK, Xie KD, Yin ZP, Wu XM, Chen CL, et al. Localization and characterization of citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling. Plant Cell Rep. 2020;39:1609–22. https://doi.org/10.1007/s00299-020-02587-z.
Xin H, Zhang T, Han Y, Wu Y, Shi J, Xi M, et al. Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides. Chromosoma. 2018;127:313–21. https://doi.org/10.1007/s00412-018-0664-y.
Yamamoto M, Takeuchi M, Nashima K, Yamamoto T. Enzyme maceration, fluorescent staining, and FISH of rDNA of pineapple (Ananas comosus (L.) Merr.) chromosomes. Horticult J. 2019;88:455–61. https://doi.org/10.2503/hortj.UTD-102.
Yu C, Deng X, Chen C. Chromosomal characterization of a potential model mini-citrus (Fortunella hindsii). Tree Genetics & Genomes. 2019;15:73. https://doi.org/10.1007/s11295-019-1379-9.
Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 2008;9:R137. https://doi.org/10.1186/gb-2008-9-9-r137.
Zhong CX, Marshall JB, Topp C, Mroczek R, Kato A, Nagaki K, et al. Centromeric retroelements and satellites interact with maize kinetochore protein CENH3. Plant Cell. 2002;14:2825–36. https://doi.org/10.1105/tpc.006106.
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Copyright (c) 2025 Dr. Elena Petrova, Dr. Kenji Tanaka, Prof. Samuel Jones
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