40. Construction of overlapping BAC clones for xa5 region of chromosome 5

A.C. SANCHEZ1, D. YANG1, G.S. KHUSH1, Y. ZHU2 and N. HUANG 1,3

1) International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines
2) Wuhan University, Wuhan, 430072 P.R. China
3) Current Address: Applied Phytologics, Inc., Sacramento, CA 95834, USA

The gene, xa5, is one of several genes for resistance to bacterial blight of rice. It confers resistance to races 1, 2, 3, and 5 and also expresses a pleiotropic effect on rate reducing resistance to race 4 of Xanthomonas oryzae pv. oryzae (Xoo) (Yoshimura et al. 1984). We report the identification of a bacterial artificial chromosome (BAC) clone carrying xa5 through fine mapping using RFLP, STS and BAC-end markers. Overlapping BAC clones spanning the xa5 region of chromosome 5 were constructed.

A genetic map of xa5 region was constructed using 180 F2 plants from the cross of new plant type line IR65598-112-2 (susceptible to Xoo) and IRBB5 (homozygous for xa5) to determine the flanking markers of the gene. RFLP markers RG556, RG207 and RZ390, previously found to be completely linked with xa5 (Yoshimura et al. 1995) were reinvestigated using this population. STS markers based on RG556 and RG207 were found to be situated at a distance of 0.8cM and 1.1cM respectively, on both sides of xa5.

A BAC library consisting of 18,432 clones and constructed from the rice cultivar IR64 (Yang et al. 1997) was screened using the three xa5-linked RFLP's. Two BAC clones 28N22 and 9E8 were identified by RZ390 for xa5. RG556 and RG207 identified five more clones e.g. 42J19, 44B4, 41N1, 7J14, and 39N11. The clone 44B4 carries both the RG556 and RG207. The two BAC clones 28N22 and 7J14 were again used to screen the library and another six clones were identified. The clones 40F20, 28P14, 4M1 and 18N18 were identified by 7J14 while 8L9 and 25P14 were identified by 28N22. Therefore, a total of 14 BAC clones were used in the construction of the xa5 BAC contig.

Ends of BAC inserts were amplified using the thermal asymmetric interlaced (TAIL) -PCR method (Liu and Whittier 1995). The PCR products from the TAIL-PCR procedure, the STS markers, the BAC clones themselves and the RFLP markers were used as probes to fingerprint the clones and order them into a contig. Finally, a contig consisting of the 14 BAC clones spanning 550kb was generated.

Two probes from the TAIL-PCR procedure generated polymorphic bands between the parents of the mapping population and were mapped relative to the other RFLP markers near xa5. This allowed us to draw a physical map of the xa5 region (Fig. 1). Other probes from the TAIL-PCR procedure did not detect polymorphism or produced multiple bands and were not used for mapping. The first polymorphic marker was derived from the right end of the BAC clone 28N22, hence, 28N22R. Linkage analysis indicated that 28N22R is about 4.7cM from xa5. Another marker, 40F20R, from the right end of 40F20, was estimated to be about 5.7cM from xa5 and 10.4cM from 28N22R. It was concluded that the BAC contig contained the xa5 locus. Since both RG556 and RG207 hybridized to BAC clone 44B4, it is concluded that the xa5 locus is within the BAC clone 44B4.


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

Yoshimura, S., A. Yoshimura, N. Iwata, S. McCouch, M. Abenes, M. Baraoidan, T.W. Mew and R.J. Nelson, 1995. Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers. Mol. Breeding 1: 375-387.

Yang, D., A. Parco, S. Nandi, P. Subudhi, Y. Zhu, G. Wang and N. Huang, 1997. Constuction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromsome 4-specific RFLP markers in rice. Theor Appl Genet 95: 1147-1154


Fig. 1. The physical map of BAC clones covering xa5 gene on the short arm of chromosome 5. The molecular markers, genetic loci, genetic distance, physical distance and total coverage on the physical map are shown on the bottom of the map. The genetic distance is based on the Southern analysis. The markers, 28N22R and 40F20R derived by TAIL-PCR from the right end of BAC clones are represented by square bar. The overlapping clones are representd by bold bar.