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. 2013 Mar 20;18(2):231–248. doi: 10.2478/s11658-013-0086-z

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

Rosa Anna Cifarelli 186, Olimpia D’Onofrio 186, Rosalba Grillo 186, Teresa Mango 186, Francesco Cellini 186, Luciana Piarulli 286, Rosanna Simeone 286, Angelica Giancaspro 286, Pasqualina Colasuonno 286, Antonio Blanco 286, Agata Gadaleta 286,✉
PMCID: PMC6275905  PMID: 23515937

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.

Key words: 5-Azacytidine, DNA methylation, Powdery mildew, Microarray, Durum wheat, Near-isogenic line, Candidate gene, Quantitative real-time PCR, Physical mapping, Pm36 gene, Expressed sequence tag

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Abbreviations used

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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