Chromosome Research

, Volume 19, Issue 5, pp 591–605 | Cite as

Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

  • Kiyotaka Nagaki
  • Fukashi Shibata
  • Go Suzuki
  • Asaka Kanatani
  • Souichi Ozaki
  • Akiko Hironaka
  • Kazunari Kashihara
  • Minoru Murata


Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.


centromere retrotransposon tobacco Nicotiana BAC chromatin immunoprecipitation 



Analysis of variance




Bacterial artificial chromosome


The basic local alignment search tool


BLAST for nucleotide


Centromere-specific histone H3


Centromeric repeat type C in maize


Centromeric tandem repeat in Oryza sativa


Clamped homogeneous electric fields


Chromatin immunoprecipitation


Centromeric retrotransposon


Centromeric retrotransposon in maize


Centromeric retrotransposon in rice


Centromeric retrotransposon in wheat






Ethylendiaminetetraacetic acid


Field inversion gel electrophoresis


Fluorescence in situ hybridization


Fluorescein isothiocianate



LB medium

Luria–Bertani medium


Long terminal repeat


2-(N-morpholino) ethanesulfonic acid


Million years ago


Nicotiana tabacum centromere-specific histone H3


Nicotiana tabacum centromeric retrotransposon


Nicotiana tabacum centromeric retrotransposon non-autonomous type


Nicotiana tomentosiformis centromeric retrotransposon

Tukey HSD

Tukey’s honestly significant difference

Pel fraction

Bound fractions in ChIP


Phenylmethylsulfonyl fluoride


Real-time quantitative PCR


Standard error


Sodium dodecyl sulfate

STE lysis buffer

SDS Tris and EDTA lysis buffer


Tris-buffered saline


Tomato genomic repeat IV


Tris (hydroxymethyl) aminomethane





This work was supported by the Program for the Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) and a grant from the Inamori Foundation (#200835).

Supplementary material

10577_2011_9219_Fig8_ESM.jpg (49 kb)
Figure S1

Multi-color FISH on SR1 chromosomes. DAPI-stained N. tabacum SR1 chromosomes (b), NtBAC#1 (c), #2 (d), #9 (e), and #10 (f) FISH signals, and merged image of b (white), c (red), d (yellow), e (green), and f (blue) (a). Scale bar, 10 μm (JPEG 48 kb)

10577_2011_9219_MOESM1_ESM.tif (6.4 mb)
High resolution (TIFF 6585 kb)
10577_2011_9219_Fig9_ESM.jpg (95 kb)
Figure S2

Immunostaining and FISH. DAPI-stained N. tabacum chromosomes (b, f and j), immunosignals of anti-NtCENH3 antibody (c, g and k), and NtBAC#2 (d), #9 (h), and #10 (l) FISH signals and merged image of bd (a), fh (e), and jl (i). Scale bar, 10 μm (JPEG 95 kb)

10577_2011_9219_MOESM2_ESM.tif (10.7 mb)
High resolution (TIFF 10971 kb)
10577_2011_9219_MOESM3_ESM.doc (116 kb)
Table S1 Primers used in this study (DOC 115 kb)
10577_2011_9219_MOESM4_ESM.doc (45 kb)
Table S2 Tobacco BAC clones showing centromeric FISH signals (DOC 45 kb)


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Kiyotaka Nagaki
    • 1
  • Fukashi Shibata
    • 1
  • Go Suzuki
    • 2
  • Asaka Kanatani
    • 1
  • Souichi Ozaki
    • 1
  • Akiko Hironaka
    • 1
  • Kazunari Kashihara
    • 1
  • Minoru Murata
    • 1
  1. 1.Institute of Plant Science and ResourcesOkayama UniversityKurashikiJapan
  2. 2.Division of Natural ScienceOsaka Kyoiku UniversityKashiwaraJapan

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