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Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

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Abstract

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.

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Abbreviations

ANOVA:

Analysis of variance

Av:

Average

BAC:

Bacterial artificial chromosome

BLAST:

The basic local alignment search tool

BLASTN:

BLAST for nucleotide

CENH3:

Centromere-specific histone H3

CentC:

Centromeric repeat type C in maize

CentO:

Centromeric tandem repeat in Oryza sativa

CHEF:

Clamped homogeneous electric fields

ChIP:

Chromatin immunoprecipitation

CR:

Centromeric retrotransposon

CRM:

Centromeric retrotransposon in maize

CRR:

Centromeric retrotransposon in rice

CRW:

Centromeric retrotransposon in wheat

DAPI:

4,6-Diamino-2-phenylindole

DIG:

Digoxigenin

EDTA:

Ethylendiaminetetraacetic acid

FIGE:

Field inversion gel electrophoresis

FISH:

Fluorescence in situ hybridization

FITC:

Fluorescein isothiocianate

IPTG:

Isopropylthio-β-d-galactoside

LB medium:

Luria–Bertani medium

LTR:

Long terminal repeat

MES:

2-(N-morpholino) ethanesulfonic acid

MYA:

Million years ago

NtCENH3:

Nicotiana tabacum centromere-specific histone H3

NtCR:

Nicotiana tabacum centromeric retrotransposon

NtCR-NA:

Nicotiana tabacum centromeric retrotransposon non-autonomous type

NtoCR:

Nicotiana tomentosiformis centromeric retrotransposon

Tukey HSD:

Tukey’s honestly significant difference

Pel fraction:

Bound fractions in ChIP

PMSF:

Phenylmethylsulfonyl fluoride

qPCR:

Real-time quantitative PCR

SE:

Standard error

SDS:

Sodium dodecyl sulfate

STE lysis buffer:

SDS Tris and EDTA lysis buffer

TBS:

Tris-buffered saline

TGRIV:

Tomato genomic repeat IV

Tris:

Tris (hydroxymethyl) aminomethane

X-gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactoside

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Acknowledgments

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).

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Authors and Affiliations

  1. Institute of Plant Science and Resources, Okayama University, Kurashiki, 710-0046, Japan

    Kiyotaka Nagaki, Fukashi Shibata, Asaka Kanatani, Souichi Ozaki, Akiko Hironaka, Kazunari Kashihara & Minoru Murata

  2. Division of Natural Science, Osaka Kyoiku University, Kashiwara, 582-8582, Japan

    Go Suzuki

Authors
  1. Kiyotaka Nagaki
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  2. Fukashi Shibata
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  3. Go Suzuki
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  4. Asaka Kanatani
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  5. Souichi Ozaki
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  6. Akiko Hironaka
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  7. Kazunari Kashihara
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  8. Minoru Murata
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Corresponding author

Correspondence to Kiyotaka Nagaki.

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Responsible Editor: Pat Heslop-Harrison.

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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)

High resolution (TIFF 6585 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 b–d (a), f–h (e), and j–l (i). Scale bar, 10 μm (JPEG 95 kb)

High resolution (TIFF 10971 kb)

Table S1

Primers used in this study (DOC 115 kb)

Table S2

Tobacco BAC clones showing centromeric FISH signals (DOC 45 kb)

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Nagaki, K., Shibata, F., Suzuki, G. et al. Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres. Chromosome Res 19, 591–605 (2011). https://doi.org/10.1007/s10577-011-9219-2

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  • DOI: https://doi.org/10.1007/s10577-011-9219-2

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