The Bacterial blight (BB) resistance gene Xa4 used to be the most widely exploited resistance gene in many Asian rice breeding programs and conferred durable resistance in many commercial rice cultivars (Mew et al. 1992). It is also a part of a resistance gene cluster including Xa3, Pi-1 (t), Pi-k, and Pi-f on chromosome 11 (Xiao et al. 1992, Leister et al. 1999). Moreover, the rice lines pyramiding Xa4 with other BB resistance genes resulted in higher resistance level and even wider resistance spectrum than those with a single resistance gene (Huang et al. 1997). Therefore, we have chosen Xa4 as a target for positional cloning.

Xa4 was first located by Yoshimura et al. (1992) on rice chromosome 11 distal to the RFLP marker G181. Li et al. (1999) mapped it between RZ536 and G2132b. Recently, several resistance gene analogues (RGAs) amplified from rice genomic DNA with degenerate primers were mapped in this region (Leister el al. 1999). In order to screen RGAs associated with Xa4 locus, we also exploited the PCR-based approaches. One of the RGAs, RSI3, with high sequence similarity to known NBS-LRR genes, had been found co-segregating with Xa4 in an F₂ population derived from an indica variety, IR24, and its Xa4 NIL, IRBB4 and located between RFLP markers G181 and L1044. Thus, RS13 could be used to land at the Xa4 locus.

In order to isolate the Xa4 gene, a BAC library consisting of 55,296 clones of IRBB56 (kindly provided by N. Huang of IRRI) was constructed (Wang et al. 2000). The markers RSI3, G181, and L1044 were used to screen the library, resulting in 18, 13, and 106 hits, respectively. Among the 18 candidate BACs detected by RS13, four (1F21, 26D24, 56M22, and 111E1) were also detected by RFLP marker G181; and six (1F21, 33M8, 56M22, 61A13, 104B15, and 111E1) were detected by L 1044. Based on the HindIII-restriction patterns, 12 of the 18 BACs were selected and digested with HindIII for Southern analysis. G181 detected one band common to 56M22, 111E1, and 26D24. Thus, G181 was located on the overlapping part of these clones. L1044 detected bands in two different positions, one band common to 56M22 and 111E1, both included G181, another common to 104B15, 33M8, and 61A13. According to the genetic distance between G181 and L1044, L1044 should be in the overlapping part of 104B15, 33M8, and 61A13. RS13 detected two to five bands each common to at least two BACs, confirming the overlapping relationship of these BACs and implying the presence of clustered homologues of RS13 in this region. The overlapping relationship of the BACs was further confirmed by Southern analysis using insert-end probes isolated from the BACs with TAIL-PCR (Liu and Whittier 1995). The insert size of each BAC was determined by CHEF electrophoresis after digestion with NotI. Subsequently, a BAC contig spanning 420 kb covering the Xa4 locus was constructed (Fig. 1). Among 339 BB susceptible individuals from the F₂ population of the Xa4 NILs, insert-end 56M22F detected 3 recombinants, 26D24R five recombinants,

and 104B15R six recombinants. Since the recombinants detected by 56M22F were different from those detected by 26D24R and 104B15R, Xa4 should be located in the region between 56M22F and 26D24R with a physical length of approximately 90 kb (Fig. 1).

Since RS13 is highly homologous to NBS domain of NBS-LRR class genes and cosegregated with the Xa4 gene, it is possible that RS13 is a part of a member in the Xa4 gene family. In a rice accession harboring Xa4, RS13 could detect 5 HindIII bands. It is noted that among the BACs detected by RS13 in Southern analysis, the clones 106P13 could display all the hybridization bands and cover the region between 56M22F and 26D24R, thus, 106P13 presumably contains Xa4 allele and was selected for further investigation.

References

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Leister, D., J. Kurth, D.A. Laurie, M. Yano, T. Sasaki, A. Graner and P. Schulze-Lefert, 1999. RFLP- and physical mapping of resistance gene homologues in rice (O. sativa) and Barley (H. vulgare). Theor. Appl. Genet. 98: 509-520.

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