Map-Based Cloning Of Rice Bacterial Blight Resistance Gene Xa23

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty-two bacterial blight resistance genes have been identified from cultivated rice and wild rice species. Six of these resistance genes, Xa1, xa5, xa13, Xa21, Xa26 and Xa27 have been cloned. It is essential to identify and clone new dominant genes with broader resistance spectrum genes for application in hybrid rice breeding. BB resistance gene Xa23, with wide spectrum resistance, complete dominance and all growth stage resistance, has been identified from O. rufipogon and it's near-isogenic line CBB23 has been developed.In this investigation, a F₂ population with 2562 individuals derived from JG30/ CBB23 was used for fine mapping and molecular cloning of Xa23;CBB23 BAC library were constructed and screened;Shotgun library of the positive BAC clone were constructed and sequenced;Candidate genes were transferred into susceptible variety for functional analysis. The main results are as following:1. Based on our previous mapping of Xa23 gene, 12 EST markers from Rice Genome Program (RGP) database were surveyed in the susceptible F₂ plants and EST markers C189 was found closely linked with the Xa23 gene, with genetic distance of 0.8cM. The marker C189 was successfully used in rice breeding program. The results showed that the efficiency of C189 marker-assisted selection (MAS) for Xa23 gene was almost 100%.2. ATAC library of Minghui 63 and a PAC library of Guangluai 4 were screened using the 0.8 kb DNA probe amplified by primer of the EST marker C189 from CBB23. Fifteen end-fragments were isolated from the positive TAC clones or PAC clones. RFLP-Southern blotting analysis showed that 6 of the 15 end-fragments displayed polymorphism between CBB23 and the susceptible recurrent parent JG30. Consequently, individual plants from F₂ population of JG30/CBB23 were further RFLP-surveyed using the 6 polymorphic end-fragments 69B, 81N, 45B, 45N, 84N and 84B as probes. The results showed that 69B, 81N, 45B, 45N, 84N and 84B were linked to the Xa23 gene with genetic distances of 0.4 cM, 0.4 cM, 0.5 cM, 0.6 cM, 0.6 cM and 1.1 cM, respectively.3. Based on the region of previous mapping of Xa23 gene and Rice Genome Program (RGP) database, we exploited 8 new PCR markers Lj13, Lj36, Lj46, Lj55, Lj204 (Lj74), Lj210, Lj211and Lj215. Individual plants from F₂ population were surveyed using these 8 markers. Linkage analysis revealed that Lj36, Lj46 and Lj211 located on the same side, with genetic distances of 0.4cM, 0.3cM and 0.2cM, respectively. The marker Lj13 located on the other side of Xa23, with genetic distance of 1.1cM. The marker Lj204 (Lj74), Lj210, Lj215 and Lj55 were co-segregated with Xa23 gene.4. CBB23-BAC library was constructed. The library contains 36,096 clones with an average insert size of 108Kb. Based on a haploid genome size of 430Mb, the coverage of the library was about 9.1 genome equivalents. The BAC library was screened using markers Lj211 and Lj74 respectively. Five positiveclones, 64F16, 71H3, 91A1, 608 and 59F8 were obtained and a contig covering the Xa23 locus was constructed. The BAC clone 608 with an insert of 80Kb was selected as the candidate gene clone.5. BAC clone 6O8 was sequenced by shotgun and a contig sequence of 72115bp was obtained. Twelve ORFs, which encode different proteins, were found by http://rgp.dna.affrc.go.ip and Softberry software. Six ORFs were selected asXa23 candidate genes.6. Ten TAC vectors containing the Xa23 candidate genes were transferred into rice cultivars Mudanjiang 8 and Zhongghua 11 by agrobacterium-mediated transformation. A total of 1350 transgenic plants were obtained and inoculated with Philippine race 6 (P6) using the leaf-clipping method. Transgenic To plants by vector T189 showed a resistance phenotype. Among 258 T189-transgenic plants, 23 were resistant to P6. Among of them, 17 transgenic plants showed high resistance (HR) and 6 showed moderate resistance (MR). The transgenic plants with other 9 vectors were all susceptible to P6. Thus, we concluded that T189 containing complete ORF6 corresponds to the function Xa23 gene.7. Further BlastX analysis showed that the ORF6 is totally different from all the previously cloned plant disease resistance genes. The Xa23 gene represents a new type of plant disease resistance genes.