Pameda C. RONALD and Steven D. TANKSLEY
Department of Plant Breeding, Cornell University, Ithaca, NY 14853, U.S.A.
Bacterial blight disease of rice caused by Xanthomonas campestris pv. oryzae (Xco) is one of the most destrucitve diseases of rice in Asia. Genetic resistance is the most effective and economical control for bacterial blight. Nineteen genes conferring resistance to specific races of Xco have been identified by classical genetic analysis. Recently, a new source of resistance was identified in the wild species of O. longistaminata (Khush et al. 1990). Unlike the other genes, Xa-21 confers resistance to all Philippine races of Xco. We have begun to characterize this locus by genetically and physically mapping the region with the goal of cloning the gene by chromosome walking techniques.
Near isogenic lines (NILs) can be used to find markers that are very close to an introgressed gene from a wild relative due to the polymorphism of such a segment in the background of a recurrent parent (Young et al. 1988). NILs for Xa-21 using recurrent parent IR24 were produced at IRRI. These NILs were surveyed with 100 rice genomic clones that were evenly distributed on the rice RFLP map (McCouch et al. 1988). One marker (RG103) that had previously been mapped to chromosome 11 was identified that showed polymorphism between the isogenic lines. This marker was tightly linked to Xa-21 in an F2 population segregating for Xa-21. Flanking markers were monomorphic on the surveys, therefore pinpointing the known introgressed interval to <20 cM on chromosome 11. We have also used random amplified polymorphic DNA (RAPD) techniques to identify primers that amplified DNA that is polymorphic between the NILs (Martin et al. 1990). We have surveyed 864 random primers (corresponding to approximately 2100 loci) and have identified 2 amplification products that are tightly linked to Xa-21. The three markers show no recombination with Xa-21 in a population of 45 plants.
In order to utilize these tightly linked markers to "walk" to Xa-21, it is necessary that the markers be very close to one another in terms of physical as well as genetic distance. Pulsed field gel electrophoresis (PFGE) of high molecular weight DNA from rice protoplasts was used to estimate the physical distance between markers. The two RAPD markers hybridize to the same 125 kbp Mlu1 fragment. These results indicate that the Xa-21 linked markers are not only tightly linked genetically, but also show close physical linkage.
Once Xa-21 is bracketed by two molecular markers and a physical map has been established, the region between these two markers can be cloned. For this purpose, we have begun construction of a yeast artificial chromosome library (Burke et al. 1987) of DNA from a rice cultivar containing the gene Xa-21.
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Martin, G., J.G.K. Williams, and S. D. Tanksley. 1990. Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and nearly isogenic lines. PNAS 88: 2336-2340.
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