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Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study

Cellular & Molecular Biology Letters volume 18, pages 231–248 (2013)Cite this article

Abstract

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

Abbreviations

5-AzaC:

5-azacytidine

BAC:

bacterial artificial chromosome

EST:

expressed sequence tags

GST:

glutathione s-transferase

HSP90:

heat shock protein

NIL:

near-isogenic line

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Authors and Affiliations

  1. Metapontum Agrobios, S.S. Jonica 106, 75010, Metaponto di Bernalda, Province of Matera, Italy

    Rosa Anna Cifarelli, Olimpia D’Onofrio, Rosalba Grillo, Teresa Mango & Francesco Cellini

  2. Department of Soil, Plant and Food Sciences, University of Bari “Aldo Moro”, Via G. Amendola 165/A, 70126, Bari, Italy

    Luciana Piarulli, Rosanna Simeone, Angelica Giancaspro, Pasqualina Colasuonno, Antonio Blanco & Agata Gadaleta

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  1. Rosa Anna Cifarelli
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  2. Olimpia D’Onofrio
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  3. Rosalba Grillo
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  4. Teresa Mango
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  5. Francesco Cellini
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  6. Luciana Piarulli
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  10. Antonio Blanco
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  11. Agata Gadaleta
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Correspondence to Agata Gadaleta.

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Cifarelli, R.A., D’Onofrio, O., Grillo, R. et al. Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study. Cell Mol Biol Lett 18, 231–248 (2013). https://doi.org/10.2478/s11658-013-0086-z

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  • DOI: https://doi.org/10.2478/s11658-013-0086-z

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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