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

Cellular & Molecular Biology Letters

Rosa Anna Cifarelli¹  Luciana Piarulli²  Teresa Mango¹  Rosalba Grillo¹  Rosanna Simeone²  Antonio Blanco²  Pasqualina Colasuonno²  Francesco Cellini¹  Agata Gadaleta²  Angelica Giancaspro²  Olimpia D’Onofrio¹ 

[1] Metapontum Agrobios, Metaponto di Bernalda, Province of Matera, Italy$$;Department of Soil, Plant and Food Sciences, University of Bari “Aldo Moro”, Bari, Italy$$

关键词: 5-Azacytidine;    DNA methylation;    Powdery mildew;    Microarray;    Durum wheat;    Near-isogenic line;    Candidate gene;    Quantitative real-time PCR;    Physical mapping;    Expressed sequence tag;   

DOI  :  10.2478/s11658-013-0086-z

学科分类：分子生物学，细胞生物学和基因

来源: Uniwersytet Wroclawski * Wydzial Biotechnologii / University of Wroclaw, Faculty of Biotechnology

【 摘 要 】

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.

【 授权许可】

Unknown