e-journal

Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization

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Abstract.
Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences
on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides
on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes.

Keywords: Coffea; FISH; meiotic chromosomes; molecular cytogenetics; physical mapping; rDNA.

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Informasi Detail

Judul Seri

AoB PLANTS

No. Panggil

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Penerbit

Oxford : Oxford University Press on behalf of the Annals of Botany Company., 2013

Deskripsi Fisik

AoB PLANTS 5: plt040; doi:10.1093/aobpla/plt040

Bahasa

English

ISBN/ISSN

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Klasifikasi

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Tipe Isi

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Tipe Media

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Tipe Pembawa

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Edisi

5: plt040

Subjek

PEMULIAAN TANAMAN

Info Detail Spesifik

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Pernyataan Tanggungjawab

agus

Versi lain/terkait

Lampiran Berkas

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