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

A.C. SANcHEZ1, L. ILAG2, D. YANG’, D.S. BRAR1, F. AUSUBEL2, G.S. KHUSH’, M. YANO3,T. SASAKI3,

N. HUANG1,4 and Z. Li1

setts General Hospital, Boston, Massachusetts 02114, USA

We report the genetic and physical mapping of the recessive gene xa13, which confers specific resistance to Philippine race 6 (PX099) 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 Gi 149, 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 Hindffi fmgerprint analysis using digested clones as probes. Additional probes for chromosome walking and contig orientation were generated by TAILPCR 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 : MR. 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, S 14003, and GI 149 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 TAILPCR 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