Plant & Animal Genome VI Conference

P29

CONSTRUCTION OF A HIGH-RESOLUTION GENETIC MAP AND A COSMID CONTIG OF THE GENOMIC REGION OF A BLAST RESISTANCE GENE, Pi-b, IN RICE

1. Rice Genome Research Program, National Institute of Agrobiological Resources / Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, 305 Japan
2. Miyagi Prefectural Furukawa Agricultural Experiment Station, Suwa, Furukawa, Miyagi, 989-61 Japan

To isolate the blast resistance gene, Pi-b, in rice by map-based cloning, a high-resolution and fine-scale linkage map of the region of the Pi-b locus was constructed. A large segregating population derived from a cross between Sasanishiki, susceptible to rice blast, and Tohoku IL9, an isogenic line of Sasanishiki with the Pi-b gene, was analyzed genetically. About 3500 susceptible individuals in the segregating population were analyzed by a pooled-sampling method using restriction fragment length polymorphism (RFLP) markers. One RFLP marker was mapped on each side of Pi-b at genetic distances of 0.04 and 0.01 cM. Three genomic clones, which were produced by sub-cloning of yeast artificial chromosome clones spanning the Pi-b locus, co-segregated with Pi-b. To physically cover the Pi-b locus, five markers tightly linked to Pi-b were used to select a cosmid library constructed using the genomic DNA of Tohoku IL9. A cosmid contig with three overlapped clones was constructed as a result. Sub-cloning and partial sequencing of a cosmid clone in the contig revealed that the cosmid contained sequences with high homology to a nucleotide-binding site (NBS) found in Arabidopsis disease-resistance genes RPM1 and RPS2. The result indicated that this region would include a candidate gene of Pi-b. The function of the candidate gene is being identified by expressed sequence analyses and transformation experiments using the cosmid clones.