36. Molecular mapping of a bacterial blight resistance gene on chromosome 8 in rice

Division of Plant Breeding, Genetics and Biochemistry, International Rice Research Institute, P. O. Box 933, 1099 Manila, Philippines

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice. Six races of the bacterium have been identified in the Philippines (Vera Cruz and Mew 1989). At least eighteen genes for resistance to specific races or clusters of races of Xoo have been identified by classical genetic analysis (Ogawa 1987; Kinoshita 1991).

A recessive gene from a cultivar "Long grain" (IRRI Acc. 35023) confers resistance to Philippine race 6 (PXO99) of Xoo. Using Long grain as the donor for the resistance gene, a near-isogenic line IR6699-5-5-4-2 was developed by backcrossing five times to the recurrent parent IR24 followed by selfing. To map the resistance gene, Long grain, IR24 and the near-isogenic line, as well as the F`2` population of 132 plants from the cross of the near-isogenic line with IR24, were studied. For bulked segregant analysis, the resistant and the susceptible bulks were prepared by mixing DNA from ten resistant plants and ten susceptiple plants of the F2 population.

RAPD [random amplified polymorphism DNA] analysis was used as an initial step for mapping the gene. From the survey of 260 random primers (Operon 10-mer kits, Operon Technologies, Inc.), we identified one primer (AC5) that amplified a polymorphic band between the resistant (Long grain, near-isogenic line and resistant bulk) and susceptible (IR24 and susceptible bulk) plants. A 0.9 kb band was amplified specifically from DNA of the susceptiple parent IR24 and the susceptible bulk. The specific band was then isolated from gels and used as a hybridization probe to blots of genomic DNA of the parents and the F`2` plants. The distance between the RAPD marker AC5-900 and the resistance gene was estimated to be 5.3 cM.

To locate the RAPD marker on an RFLP map, a mapping population of 135 doubled haploid lines generated from the cross IR64/Azucena at IRRI was used. The results indicated that the RAPD marker is located between RFLP markers RZ66 and CDO99 on chromosome 8 (Fig. 1A).

Fig. 1. Partial RFLP maps of chromosome 8 of rice. The distance between markers is shown in centimorgans on the left. A. This map was developed using the mapping population of 135 doubled haploid lines derived from the cross IR64/Azucena. The AC5-900 marker is located between RFLP markers RZ66 and CDO99. B. This map was developed using the F`2` population of 132 plants derived from the cross IR6699-5-5-4-2/IR24, showing the location of the new gene for resistance to bacterial blight and closely linked markers.

For fine mapping of the resistance gene, ten RFLP markers near the RAPD marker were selected to survey the parents. Through the use of 12 restriction enzymes, DNA polymorphism between parents for RZ28 and CDO116 was detected when the DNA was digested with XhoI. RG136 showed polymorphisms when DNA was digested with BglI, DraI, EcoRI, ScaI, XbaI and XhoI. The polymorphic RFLP markers were then used to survey the filters of 132 F`2` plants. No recombinants were found between RZ28 and CDO116. The distance between these two RFLP markers and the resistance gene was estimated to be 5.1 cM. RG136 is on the other side of the gene and its distance from the gene was estimated to be 3.8 cM (Fig. 1B).

This new recessive resistance gene showed the same type of resistance to the bacterial blight as the recessive resistance gene xa-13, which was reported to be linked with another resistance gene xa-5 through classical genetic analysis (Ogawa et al. 1987). Yoshimura et al. (1984) located xa-5 on chromosome 5 through trisomic analysis. This location was confirmed by RFLP analysis by McCouch et al. (1991). We serveyed all 38 available RFLP markers on chromosomes 5, but failed to identify any linkage between these markers and the new resistance gene. Allele tests between the new gene and xa-13 are underway. A gene symbol will be assinged to this gene if it turns out to be non-allelic to xa-13.

References

Kinoshita, T., 1991. Report of the committee on gene symbolization, nomenclature and linkage groups. RGN 8: 2-37.

McCouch, S. R., M. P. Abenes, R. Angeles, G. S. Khush and S. D. Tanksley, 1991. Molecular tagging of a recessive gene, xa-5, for resistance to bacterial blight of rice. RGN 8: 143-145.

Ogawa, T., 1987. Gene symbols for resistance to bacterial leaf blight. RGN 4: 41-43. Ogawa, T., L. Lou, R. E. Tabien and G. S. Khush, 1987. A new recessive gene for resistance to bacterial blight of rice. RGN 4: 98-100.

Vera Cruz, C. M. and T. M. Mew, 1989. How variable is Xanthomonas campestris pv. oryzae? Pages 153-166 in Bacterial blight of rice. International Rice Research Institute, P. 0. Box 933, Manila, Philippines.

Yoshimura, A., T. M. Mew, G. S. Khush and T. Omura, 1984. Genetics of bacterial blight resistance in a breeding line of rice. Phytopathology 74: 773-777.