1.1.20
MOLECULAR IDENTIFICATION OF THE STRIPE RUST GENE YR10

A LAROCHE, R HUEL, M FRICK, C NYKIFORUK and R CONNER

Agriculture and Agri-Food Canada, Lethbridge Research Centre, PO Box 3000, Lethbridge T1J 4B1, Canada

Background and objectives
Stripe rust, caused by the fungus Puccinia striiformis, is one of the most serious diseases of irrigated spring wheat in western Canada. Under cool, wet conditions that are favourable for disease development, severe leaf and head infection can cause over 40% loss in yield. In addition to yield loss, grain from heavily infected fields is downgraded because of shrivelled kernels. Mutational changes in the virulence of the stripe rust pathogen have been observed in the past. Recent models show that pyramiding different disease-resistance genes within a cultivar is an effective way to ensure long-lasting resistance in wheat and other cereals [1].

Resistance genes in plants have been identified, based on plant phenotype, since the beginning of the 20th century [2]. The molecular identification of the first resistance gene, the tomato Pto gene conferring resistance to Pseudomonas syringae, was reported in 1993 [3]. Since then, less than a dozen plant disease-resistance genes have been reported, and it is expected that many more will be described in the next few years. The gene-for-gene interaction model between plant and pathogen implies that for a plant resistance reaction, the plant must recognize a specific gene product expressed by the invading pathogen. Most of the identified disease-resistance genes involve a cellular hypersensitive response leading to cell death to stop the invading pathogen. Disease-resistance genes are extracellular, intracellular, or have domains on both side of the plasma membrane. Our current interest in characterizing disease-resistance genes in wheat rests on (i) a better understanding of the interaction between plant and pathogen; (ii) a better understanding of the recognition region of the resistance gene; and (iii) the rapid identification of related genes and transfer into cultivars by conventional crossing and progeny analysis using marker-assisted selection or by transforming a wheat cultivar directly. Our primary objective was to identify Yr10 in wheat which is an effective stripe rust resistance gene in western Canada.

Materials and methods
DNA was isolated from BC3 lines of Moro (Yr10) and Fielder (susceptible), and bulks were made with lines either resistant or susceptible to stripe rust. A total of 119 RAPD primers were used to screen the two bulks. A segregating population of 133 seedlings was used to verify linkage of markers. A cDNA library was made using LamdaZapII, while a subpopulation of genomic DNA hybridizing to the Yr10 gene probe was cloned into the same vector. Putative identity of DNA fragments was verified using sequence homology.

Results and conclusions
A polymorphic DNA fragment of 1100 bp was identified only in the resistant bulked DNA fraction, and was present in all individual DNA used to make this bulk. This fragment appears to be putatively linked to the stripe rust resistance gene Yr10 present in the wheat cultivar Moro. This polymorphic fragment was perfectly linked in an F2 population of 133 lines segregating for Yr10. Upon characterization of this 1100-bp fragment, a large portion of it showed a very significant level of homology at the amino acid level (127, P 4-10) with the P-loop region of the L6 rust resistance gene in flax [4]. Furthermore, a kinase-2 domain downstream of the P-loop was identified as predicted by the flax L6 and tobacco N resistance genes, thus confirming the NBS region of the 1100-bp fragment. Like the L6 and N genes, this region of the Yr10 wheat fragment is relatively rich in leucine. Work is in progress to characterize cDNAs and genomic clones in order to identify the full-length sequence of this gene. The sequence of the different domains of this wheat stripe rust resistance gene will be discussed in relation to other intracellular resistance genes characterized in dicots.

References
1. Zwer PK, Qualset CO, 1994. Euphytica 74, 109-115.
2. Jones DA, Jones JDG, 1997. Adv. Bot. Res. 24, 89-167.
3. Martin GB, Brommonschenkel SH, Chunwongse J et al., 1993. Science 262, 1432-1436.
4. Lawrence GL, Finnegan EJ, Ayliffe MA, Ellis JG, 1995. Plant Cell 7, 1195-1206.