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Chromosoma

, Volume 117, Issue 5, pp 445–456 | Cite as

Structure and dynamics of retrotransposons at wheat centromeres and pericentromeres

  • Zhao Liu
  • Wei Yue
  • Dayong Li
  • Richard R.-C. Wang
  • Xiuying Kong
  • Kun Lu
  • Guixiang Wang
  • Yushen Dong
  • Weiwei Jin
  • Xueyong Zhang
Research Article

Abstract

Little is known of the dynamics of centromeric DNA in polyploid plants. We report the sequences of two centromere-associated bacterial artificial chromosome clones from a Triticum boeoticum library. Both autonomous and non-autonomous wheat centromeric retrotransposons (CRWs) were identified, both being closely associated with the centromeres of wheat. Fiber-fluorescence in situ hybridization and chromatin immunoprecipitation analysis showed that wheat centromeric retrotransposons (CRWs) represent a dominant component of the wheat centromere and are associated with centromere function. CRW copy number showed variation among different genomes: the D genome chromosomes contained fewer copies than either the A or B genome chromosomes. The frequency of lengthy continuous CRW arrays was higher than that in either rice or maize. The dynamics of CRWs and other retrotransposons at centromeric and pericentromeric regions during diploid speciation and polyploidization of wheat and its related species are discussed.

Keywords

Bacterial Artificial Chromosome Long Terminal Repeat Bacterial Artificial Chromosome Clone Bacterial Artificial Chromosome Library Pericentromeric Region 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

The authors are grateful to Dr. J. Jiang (University of Wisconsin, Madison, USA) for providing the RCS1 plasmid and valuable comments on the manuscript, to Dr. S. Henikoff (Fred Hutchinson Cancer Research Center) for the rice CENH3 antibody, to Dr. C. Feuillet (INRA, Clermont-Ferrand, France) for providing the 3B centromere-associated BAC clones, to J. Wu (ICS, CAAS) for help with the BAC sequencing and bioinformatic analysis, and to Z. Cheng and L. Mao (ICS, CAAS) for valuable discussion. They also thank www.smartenglish.co.uk for linguistic advice in the preparation of this manuscript. This research was supported by the Natural Science Foundation of China (39870494, 30771208).

Supplementary material

412_2008_161_MOESM1_ESM.ppt (36 kb)
Fig. S1 CRW LTRs in BAC clone TbBAC30 (5→3′ the direction of the LTR, asterisks the inverted repeat at the terminals of the LTR) (PPT 36.0 KB)
412_2008_161_Fig1_ESM.gif (30 kb)
Fig. S2

Chromosome localization of the CR2–1 sequence (part of the LTR region of an autonomous CRW) in hexaploid wheat. a Biotin-labeled probe (red). b Probed with digoxingenin-11-dUTP-labeled pAs1 (yellow/green) and biotin-16-dUTP-labeled pSc119.2 (red). The signals on the A genome centromeres are stronger than those on the B or D genome centromeres. Bars 10 μm (GIF 4.94 MB)

412_2008_161_Fig1_ESM.tif (4.9 mb)
High resolution image file (GIF 30.1 kb)
412_2008_161_Fig2_ESM.gif (77 kb)
Fig. S3

In situ hybridization in hexaploid wheat cv. Chinese Spring with amplicons derived from A. speltoides (a) or A. tauschii (b) and centromeric-associated BAC clones derived either from cv. Chinese Spring chromosome 3B (c) or A. tauschii (d) as probes. LTRm primers were used for PCR (listed in Table S4). The BAC used in c was 3B-078-D03 (centromeric signals: green) and in d was 5C01 (centromeric signals: red). Digoxingenin-11-dUTP-labeled pAs1 (yellow/green) and biotin-16-dUTP-labeled pSc119.2 (red) were used simultaneously to distinguish the chromosomes of the B and D genomes. The signals on D genome centromeres are weaker than those on A or B genome centromeres. Bars 10 μm (GIF 8.45 MB)

412_2008_161_Fig2_ESM.tif (8.5 mb)
High resolution image file (GIF 60.0 kb)
412_2008_161_MOESM6_ESM.ppt (103 kb)
Fig. S4 Representative quantitative real-time PCR as used in ChIP analysis, for determining the relative fold enrichment (RFE) of CENH3-associated sequences in the bound fraction over the mock control. All samples showed obvious enrichment of precipitated DNA, except for Erika and 5.8S. The red and green curves correspond to the two mock DNA samples (pellet), whereas the blue and yellow curves correspond to the two bound DNAs (pellet). (a) 365–1c, (b) 5′-UTR, (c) gag, (d) RT, (e) INT, (f) non-autonomous CRW LTR, (g) Erika, (h) 5.8S (CK). The cycle threshold (CT) was taken with the baseline of fluorescence intensity being manually set at a value between 0.01 and 0.05. (PPT 103 KB)
412_2008_161_MOESM7_ESM.ppt (241 kb)
Fig. S5 Southern hybridization profiles of Triticeae species, following probing with three retrotransposon sequences. (a) Wgel_TbBAC5–1p, (b) Erika_TbBAC5–1, (c) Sukkula_TbBAC5–1 with the probes of C04–1, G10, and Sukkula (see Fig. 1a and Table S4). Genome symbols are used to identify the lanes after gel electrophoresis (Cu: A. umbrellulata, Au: T. urartu, Ab: T. monococcum subsp. aegilopoides, S: A. speltoides var. ligustica, D: A. tauschii, AB: T. orientale, AG: T. araraticum, ABD: T. aestivum). The genomic DNA was fully digested by HindIII (a, b) or BamHI (c). The signals in the five species containing the A genome are significantly stronger than those in the other species. The polyploid wheats thus presumably inherited the hybridization patterns of their diploid ancestors. (PPt 241 KB)
412_2008_161_Fig3_ESM.gif (60 kb)
Fig. S6

In situ hybridization in four Triticum species, generated by probing with Erika_TbBAC5–1. (a) T. urartu, (b) T. dicoccoides, (c) T. araraticum, (d) T. aestivum. The probe sub-clone G10, encompassing a partial LTR and a partial coding region of Erika_TbBAC5–1 (marked in Fig. 1a), was labeled with digoxingenin-11-dUTP and detected with anti-digoxigenin-fluorescein (yellow/green). Chromosomes were counter-stained with propidium iodide (PI) in Vectashield for anti-digoxigenin-fluorescein detection. Dispersed signals were distributed on the arms of the A genome chromosomes and almost absent in the centromeres, showing that Erika_TbBAC5–1 is non-centromeric sequence-specific (or abundant) in the A genome (arrows in b-d translocations between the A and B or G genome chromosomes). Bars 10 μm (GIF 76.6 KB)

412_2008_161_Fig3_ESM.tif (15.6 mb)
High resolution image file (GIF 15.6 MB)
412_2008_161_MOESM10_ESM.doc (33 kb)
Table S1 Non-centromere-specific retrotransposons present in BAC clone TbBAC30 (DOC 33.0 KB)
412_2008_161_MOESM11_ESM.doc (32 kb)
Table S2 Incomplete CRWs present in BAC clones TbBAC5 and TbBAC30 (DOC 32.5 KB)
412_2008_161_MOESM12_ESM.doc (32 kb)
Table S3 The CRW, PrBS (primer-binding site), and PPT (polypurine tract) present in BAC clones TbBAC5 and TbBAC30 (DOC 31.5 KB)
412_2008_161_MOESM13_ESM.doc (64 kb)
Table S4 Probes used for Southern hybridization, FISH, and fiber-FISH (DOC 64.0 KB)
412_2008_161_MOESM14_ESM.doc (38 kb)
Table S5 Primers used for ChIP analyses (no a non-autonomous) (DOC 38.0 KB)
412_2008_161_MOESM15_ESM.doc (45 kb)
Table S6 Quantification of real-time PCR products in ChIP analyses (no a non-autonomous) (DOC 45.0 KB)

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Zhao Liu
    • 1
  • Wei Yue
    • 1
  • Dayong Li
    • 1
  • Richard R.-C. Wang
    • 2
  • Xiuying Kong
    • 1
  • Kun Lu
    • 1
  • Guixiang Wang
    • 3
  • Yushen Dong
    • 1
  • Weiwei Jin
    • 3
  • Xueyong Zhang
    • 1
  1. 1.Key Laboratory of Crop Germplasm & Biotechnology, MOA, Institute of Crop Sciences, Chinese Academy of Agricultural SciencesThe National Facility for Crop Gene Resources and Genetic ImprovementBeijingPeople’s Republic of China
  2. 2.USDA-ARS Forage and Range Research LaboratoryLoganUSA
  3. 3.National Maize Improvement Center of China, Key Laboratory of Crop Genetic Improvement and Genome of Ministry of AgricultureChina Agricultural UniversityBeijingPeople’s Republic of China

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