Molecular Mapping And Genomic Library Construction For Bacterial Blight Resistance Gene Xa23 In Rice

The rice bacterial blight (BB) resistance gene, Xa23, was originally identified from Oryza rufipogon based on the resistance spectrum evaluation, resistance type and inheritance analysis. It is a complete dominant resistance gene with the widest resistance spectrum among all the known BB resistance genes identified so far. To clone Xa23 gene, a F₂ population including 2562 plants was constructed from a pair of near-isogenic line JG30/CBB23 and Xa23 was primarily mapped on the long arm of rice chromosome 11.This research is aiming at fine mapping and cloning of Xa23 and the following results have been obtained:1. RFLP analysis was performed with 15 probes located within the region between G257 and G1465 on the long arm of rice chromosome 11. Six probes show polymorphism between JG3O and CBB23 and among of them, C1003 A is closely linked with Xa23 and the genetic distance is 0.4cM.2. The RFLP marker C1003A was transformed into sequence-tagged-site (STS) marker, which can be easily applied for molecular marker-assisted selection (MAS) of Xa23 in rice breeding programs.3. A TAC genomic library of CBB23 harboring Xa23 gene was constructed using TAC binary vector pYLTAC68HB. The library consists of 68736 individual clones. The average insert size is 32kb. The genome coverage of the TAC library was about 5.1 haploid genome equivalents of rice.4. The contig of isogenetic locus of Xa23, based on Nipponbare sequence, was constructed by BLAST with C1003A sequence and primers of SSR marker RM206. Thirty-one pairs of special primers were designed based on the contig sequence and used for amplifying fragment length polymorphism between JG30 and CBB23. Four markers, A89a2, AK601, CF20 and A83b4, were developed. Linkage analysis showed that A89a2 and AK601 was located on the same side as C1003A, with genetic distances to Xa23 of 0.4cM and 0.2cM, respectively;CF20 and A83b4 were located on the other side of Xa23, with genetic distances of 0.2cM and 0.3cM, respectively.5. Another F₂ population with 1076 individuals derived from the cross of IR24/CBB23 was constructed. The segregation of resistant and susceptible plants in the F₂ population agreed with a 3:1 ratio according to the reaction to race 6 of Xoo and the F₂ population was appropriate for mapping Xa23 gene. It is confirmed that Xa23 was located between markers STS03 and A83b4 by examining all the individuals of the population with STS03 and A83b4. Because JG30 have different genetic background with IR24, primers showing no polymorphism between JG30 and CBB23 were further analyzed for polymorphism between IR24 and CBB23. Four closer markers, A8A, A8C, CF6 and LJ478, were developed and no recombinants were found between the markers and Xa23 gene. F₃ populations from F₂ recombinants between STS03 and A83b4 were inoculated with race P6 of Xoo and the segregation of susceptible and resistant plant agreed with expected ratio.6. Two subclone libraries of BAC clone 608 were constructed with binary vectors, pCAMBIA1300 and pYLTAC747H respectively. The libraries with pCAMBIA and pYLTAC were consisted of 396 and 200 clones, respectively and the insert size ranged at 6-12kb and 10-25kb, respectively.7. Gene prediction of BAC 6O8 was performed with RiceGAAS system. Sequence analysis of thisregion revealed that ORF4, 5, 6, 7 and 8 could be the candidates of Xa23 gene. The pCAMBIA1300 transformation constructs respectively containing ORF4, 5, 7 and 8 including 5'promoter and 3'UTR, were screened out. But no constructs with complete ORF6 was obtained due to too big size of ORF6 for pCAMBIA1300. 1180 transgenic plants were generated by agrobacterium-mediattd transformation with constructs containing ORF4, 5, 7 and 8 respectively, but all the transplants showed susceptible to race P6 of Xoo.8. Seven RNAi constructs of ORF4, 5, 6, 7 and 8 were constructed with RNAi vector pCK303 and 3 of the RNAi constructs were for ORF6. These RNAi constructs had been used to transform CBB23 and Zhongyel9 harboring Xa23 gene, and some regenerated plants are prepared for inoculation with race P6 of Xoo.