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Localization and characterization of Citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling

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The physical locations of citrus centromere are revealed by combining genetic and immunological assays for the first time and nine citrus centromere-specific markers for cytogenetics are mined.

Abstract

Centromere localization is challenging, because highly redundant repetitive sequences in centromeric regions make sequence assembly difficult. Although several citrus genomes have been released, the centromeric regions and their characteristics remain to be elucidated. Here, we mapped citrus centromeres through half-tetrad analysis (HTA) that included the genotyping of 54 tetraploid hybrids derived from 2n megagametophytes of Nadorcott tangor with 212 single nucleotide polymorphism (SNP) markers. The sizes of centromeric regions, which estimated based on the heterozygosity restitution rate pattern along the chromosomes, ranged from 1.12 to 18.19 Mb. We also profiled the binding sequences with the centromere-specific histone variant CenH3 by chromatin immunoprecipitation sequencing (ChIP-seq). Based on the positions of the top ten CenH3-enriched contigs, the sizes of centromeric regions were estimated to range from 0.01 to 7.60 Mb and were either adjacent to or included in the centromeric regions identified by HTA. We used DNA probes from two repeats selected from the centromeric regions and seven CenH3-binding centromeric repeats to verify centromeric locations by fluorescence in situ hybridization (FISH). Centromere localization in citrus will contribute to the mining of centromeric/pericentromeric markers, thus to facilitate the rapid identification of mechanisms underlying 2n gamete formation and serve the polyploidy breeding.

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Funding

This research was financially supported by the National Key R&D Program of China (2018YFD1000200), the National Natural Science Foundation of China (Grant nos. 31820103011, 31701873, 31530065), and the Fundamental Research Funds for the Central Universities of China (Grant no. 2662018PY007). The authors thank our colleague Prof. Robert M. Larkin for critical reading of the manuscript.

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Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, China

    Qiang-Ming Xia, Kai-Dong Xie, Zhao-Ping Yin, Xiao-Meng Wu & Wen-Wu Guo

  2. College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Lu-Ke Miao & Chun-Li Chen

  3. Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

    Jude W. Grosser

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  1. Qiang-Ming Xia
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  2. Lu-Ke Miao
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  3. Kai-Dong Xie
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  4. Zhao-Ping Yin
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  5. Xiao-Meng Wu
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  6. Chun-Li Chen
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  7. Jude W. Grosser
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  8. Wen-Wu Guo
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Contributions

KDX and WWG conceived of the study and designed the research. QMX conducted most of the experiments and the data analysis. QMX and KDX wrote the manuscript. LKM participated in cytological verification of the centromeric repeats. ZPY participated in the bioinformatics analysis. CLC provided the anti-CsCenH3 antibody. XMW provided suggestions on experimental design and revised the manuscript. JWG provided pollen of some citrus allotetraploids. All the authors read and approved the final version of the manuscript.

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Correspondence to Kai-Dong Xie.

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Xia, QM., Miao, LK., Xie, KD. et al. Localization and characterization of Citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling. Plant Cell Rep 39, 1609–1622 (2020). https://doi.org/10.1007/s00299-020-02587-z

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