Chromosome Research

, Volume 21, Issue 4, pp 375–381 | Cite as

Tobacco karyotyping by accurate centromere identification and novel repetitive DNA localization

  • Fukashi Shibata
  • Kiyotaka Nagaki
  • Etsuko Yokota
  • Minoru Murata
Article

Abstract

Tobacco (Nicotiana tabacum) is an amphidiploid species (2n = 4x = 48, genome constitution SSTT) derived from a natural hybrid between Nicotiana sylvestris (2n = 2x = 24, SS) and Nicotiana tomentosiformis (2n = 2x = 24, TT). Genomic in situ hybridization (GISH), using the genomic DNA from these ancestral species as probes, revealed the chromosomal origins (S or T) and the occurrence of intergenomic translocations in N. tabacum. Fluorescence in situ hybridization (FISH) was also used to distinguish between chromosomes. However, the use of repetitive DNA sequences as probes for FISH analysis is limited by an inability to identify all chromosomes. In addition to this limitation, the occurrence of chromosomal tertiary constrictions can easily lead to the misclassification of chromosomes. To overcome these issues, immunostaining with anti-N. tabacum centromere-specific histone H3 antibody was carried out to determine the centromere position of each chromosome, followed by FISH analysis with ten distinct repetitive DNA probes. This approach allowed us to identify 22 of the 24 chromosome pairs in N. tabacum and revealed novel intergenomic chromosome rearrangements and B-chromosome-like minichromosomes. Hence, the combination of immunostaining with FISH and GISH is critical to accurately karyotype tobacco.

Keywords

Nicotiana tabacum fluorescence in situ hybridization (FISH) genome in situ hybridization (GISH) immunostaining centromere-specific histone H3 (CENH3) 

Abbreviations

CENH3

Centromere-specific histone H3

DAPI

4,6-diamino-2-phenylindole

DIG

Digoxigenin

FISH

Fluorescence in situ hybridization

GISH

Genomic in situ hybridization

NtCENH3

Nicotiana tabacum centromere-specific histone H3

PBS

Phosphate-buffered saline

Notes

Acknowledgment

This work was supported by the program for the promotion of basic and applied researches for innovations in bio-oriented industry (BRAIN).

Supplementary material

10577_2013_9363_Fig3_ESM.jpg (24 kb)
Fig. S1

A GISH image of N. tabacum cv. Petit Havana SR1. A mitotic prometaphase cell labeled by GISH, using genomic DNA from N. tomentosiformis (green) and N. sylvestris (red). Scale bar, 2 μm. (JPEG 24 kb)

10577_2013_9363_MOESM1_ESM.tif (1.5 mb)
High-resolution image (TIFF 1,560 kb)
10577_2013_9363_MOESM2_ESM.doc (146 kb)
Table S1 (DOC 146 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Fukashi Shibata
    • 1
  • Kiyotaka Nagaki
    • 1
  • Etsuko Yokota
    • 1
  • Minoru Murata
    • 1
  1. 1.Institute of Plant Science and ResourcesOkayama UniversityKurashikiJapan

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