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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Analysis of Four Resistance Gene Analogs from Wheat Using Conserved Primers

Authors
item Goodwin, Stephen
item Hu, Xueyi - USDA-ARS

Submitted to: Wheat Genetics International Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: April 16, 1998
Publication Date: N/A

Interpretive Summary: Fungal diseases of wheat cause hundreds of millions of dollars in losses to United States cereal growers every year. Breeding for increased resistance could be accelerated greatly with better genetic characterization of resistance genes. Recently, a number of disease resistance genes from plants have been cloned. Analyses of the DNA sequences showed that certain regions were highly conserved among resistance genes from very diverse plants. The conserved regions of known resistance genes can be used to clone similar sequences from other plant species. These sequences are called resistance gene analogs because they are similar to previously studied resistance genes and may in fact be resistance genes themselves. To test the hypothesis that similar sequences occur in wheat, regions corresponding to resistance gene analogs were cloned from a wheat line with a gene for resistance to the fungal disease powdery mildew. Approximately 300 clones were obtained and divided into 10 classes. DNA sequence analysis showed that the 10 classes could be reduced to only four, and all had the characteristics of previously cloned resistance genes. Database searches with the four clones showed that they were closely related to previously identified resistance gene analogs from barley and rice. Experiments are now under way to uniquely identify each of the four resistance gene analog classes, and to determine if any is linked to known disease resistance genes in wheat. This information will be useful to plant pathologists and geneticists studying disease resistance in wheat, and ultimately will lead to a better understanding of plant disease resistance genes.

Technical Abstract: Degenerate primers based on the conserved nucleotide binding sequence (NBS) of the cloned plant disease resistance genes N, L6 and RPS2, were used to amplify genomic DNA of two wheat lines that were near-isogenic for a powdery mildew resistance gene. Both lines gave a diffuse band of approximately 500 base pairs, and one or two additional larger bands. Amplification products from the resistant line in the 500 base-pair size range were extracted from agarose gels and cloned using a TA cloning kit. Following color selection, clones containing inserts of approximately 500 base pairs were identified by digesting plasmid DNA with the restriction enzyme Eco RI and separating the fragments by gel electrophoresis. Restriction analysis of approximately 300 randomly chosen clones using four enzymes with 4- or 6-base recognition sequences separated the putative resistance gene analogs into at least 10 different classes. Sequence analysis of representative clones from each of the 10 resistance gene analog classes and analyses of their probable translation products revealed that all encoded the same conserved motifs found in the cloned plant disease resistance genes N, L6 and RPS2. Thus, each class represented the nucleotide binding sequence of a resistance gene analog. Specific primers are being constructed from the variable regions to provide specific amplification of each RGA class. These primers then will be used to determine whether any of these RGA sequences is linked to any of the known genes for powdery mildew resistance, and to estimate the probable map location and linkage relationships of each RGA from wheat.

Last Modified: 10/25/2014
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