29. Identification of overlapping BAC clones carrying the xa13 locus in rice

A.C. SANCHEZ1, L. ILAG2, D. YANG1, D.S. BRAR1, F. AUSUBEL2, G.S. KHUSH1, M. YANO3,T. SASAKI3,N. HUANG1,4 and Z. LI1

1) International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines
2) Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
3) Rice Genome Research Program, NIARJSTAFF, Tsukuba, Ibaraki, 305-8602 Japan
4) Applied Phytologics Inc., Sacramento, California 95834, USA

     We report the genetic and physical mapping of the recessive gene xa13, which confers specific resistance to Philippine race 6 (PXO99) of Xoo. Resistance conferred by recessive genes like xa13 may represent very different and largely unknown biochemical pathways in the host defense system. Nine selected DNA markers and two F2 populations (NIL cross n = 131 and NPT cross n = 230) were used to construct a genetic map of the xa13 region of rice chromosome 8. Four DNA markers, RG136, R2027, S14003 and G1149, closely flanking xa13 were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library constructed using IR64 (Yang et al. 1997). All candidate BAC clones identified by colony hybridization were subjected to Southern hybridization analysis to confirm their overlaps. Clones were built into contigs based on HindIII fingerprint analysis using digested clones as probes. Additional probes for chromosome walking and contig orientation were generated by TAIL-PCR of the positive BAC clones detected from colony hybridization (Liu and Whittier 1995).
     Clear and distinct reactions to the Xoo race 6 were observed among the parental lines and F2 plants. In the NIL F2 population, plants segregated into 106 susceptible (S) and 25 resistant (R) while in the NPT F2 population, the segregation was 166S : 64R. both agreeing with the expected 3:1 Mendelian ratio. All RFLP markers in the xa13 region also agreed with the expected ratio of 1:2:1 at the 5% level. Figure 1 shows the two genetic maps containing xa13 and all the selected DNA markers used in the two mapping populations. The linear order of RG136, R2027, R2662, S14003, and G1149 in our maps was consistent with that determined by Harushima et al. (1998). Our results clearly indicated that of these markers, RG136 and R2027 were the closest ones flanking the xal3 locus. Table 1 shows the probes used to screen the IR64 BAC library by colony hybridization and the corresponding positive BAC clones. Overlaps were confirmed by hybridization of the positive clones with the RFLP markers. Insert ends of the identified BAC clones were amplified by TAIL-PCR and used as probes for hybridization with each clone in the contigs. The resulting alignment of the overlapping clones in these contigs was determined based on DNA hybridization patterns using the digested clones and TAIL-PCR products as probes.

     A single copy BAC end marker 42C23R, derived from the right end of 42C23, was mapped 1.2 cM from xa13 while R2027 was 1 cM on the other side using the NPT population. Another BAC-derived marker, 6B7F, is 0.8 cM beyond R2027. Two additional markers, 21H14F (left end) and 21H14R (right end), derived from clone 21H14, were found to flank xa13 on either side using the NIL population. These results indicated that the contig and clone 21H14 contained the xa13 locus. The genetic distance between the insert ends of 2lHl4 was determined to be 1 cM while the physical size of 21H14 insert is -96 kb, giving a physical/genetic ratio of 96 kb/cM in the xa13 region of chromosome 8. Figure 2 shows the genetic map of xa13 aligned with the physical map.
     The construction of a contig map with overlapping BAC clones encompassing the xa13 locus represented a significant step toward our final goal to isolate this gene. Our results indicated that in the xa13 region of chromosome 8, each cM of genetic distance is roughly equivalent to 96 kb, a nearly 3-fold reduction in the ratio of physical/genetic map as compared to the average ratio of 260-280 kb/cM estimated by Wu and Tanksley (1993) and by Harushima et al. (1998). This reduction was apparently due to enhanced recombination in the region. The following steps to isolate xa13 including construction of the xa13 cDNA library, identification of the positive cDNA clones for the resistant allele of xal3, sub-cloning of clone 21H14, genetic complementation, etc., are underway.

References

Harushima, Y., M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki,T. Yamamoto, S.Y. Lin, B.A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G.S. Khush and T. Sasaki, 1998. A high density rice genetic linkage map with 2275 markers using a single F2 population. Genetics 148: 479-494.

Liu, Y.G. and R.F. Whittier, 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674-681.

Wu, K.S. and S.D. Tanksley, 1993. PFGE analysis of the rice genome: estimation of the fragment sizes, organization of the repetitive sequences and relationships between genetic and physical distances. Plant Mol. Biol. Rep. 23: 243-254.

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