| Bacterial blight, caused by Xanthomonas oryzae pv. Oryzae, seriously reduces rice production world-wide. Two bacterial blight resistance genes, Xa3 and Xa4, were concentratively used for many years in China. It is very necessary to utilize new bacterial blight resistance genes to improve the resistance of target varieties. Xa22(t), identified from Zhachanglong(ZCL), conferred high levels of adult resistance to all 12 bacterial blight strains tested. Xa26, cloned from Minghui63-BAC clone named M3H8, had identical alleles in IRBB3 and ZCL, furthermore Xa3 and Xa26 may be the same gene and ZCL contained Xa26.(1) Fine mapping of Xa22(f)p2 population of 7680 plants derived from the cross of Zhenzhu'ai and Zhachanglong was screened for recombination events by RM144 and RM224. In all, 29 susceptible recombinants plants were identified by RM144 and RM224 after inoculation with PXO61 at the booting stage. Among the 29 susceptible recombinants plants, 20 were derived from recombination events between Xa22(i) and RM144 and 9 between Xa22(i) and RM224. Two recombination events were identified between Xa22(i) and 3/7A10 (one subclone of M3H8), and one recombinant was identified between Xa22(t) and R1506. In addition, both 3/7A10 and R1506 hybridized to BAG clone M3H8, this indicated that the Xa22(t) locus resided on this 100-kb BAC clone, M3H8.One Nipponbare sequence clone named AC116367 was identified by Xa26 sequence. Further analysis showed that many resistance gene analogs. In combination with the map position of AC116367 in Xa22(i) region, Nipponbare AC116367 was regarded as the electronic map clone of Xa22(i).(2) Candidates ofXa22(t)Restriction enzyme maps of two fragments in M3H8 were analyzed and equipotential digestion was used to recover the corresponding fragments from ZCL. A fragment of 19-kb digested by Spel contained Xa26 and a fragment of 8-kb digested by Nhel recovered LRR, while 12-kb fragment digested byAccl contained LRR and Kinase. After digestion, fragments of the expected size were recovered and verified by specific primers. Then the expected fragments were cloned into intermediate vector and confirmed by PCR further. Finally, the target fragment was transferred to pCAMBIA1301 for transformation. Now, 10 transformants ofXa26 and one transformant of LRR containing fragment showed resistance to PXO61. For each transformation mentioned, about 100transformants of each fragment grew up.One ZCL-BAC clone named 80112 was identified and a 12-kb fragment of it hybridized with R1506, and the 12-kb fragment proved containing NBS. Further analysis showed that M3H8 contained NBS and NBS was expressed in ZCLby RT-PCR with NBS amplification. Finally, one transformant of this 12-kb fragment conferred resistance to PXO61.(3) RT-PCR expression analysisThe RT-PCR expression of Xa26, LRR, Kinase and RKORF1 was assayed in three resistant varieties ZCL (Xa22(i)\ Wase Aikoku 3 (WA3, Xa3) and IRBB4 (Xa4).Primers in Xa26 and RKORF1 were used to carry out RT-PCR analysis. Xa26 was constitutively expressed in above three resistant varieties, while no expression was detected in ZZA. RKORF1 identified the same result as Xa26.NBS was constitutively expressed in ZCL and IRBB4, while not in WA3, which contained Xa3. This indicated that NBS-LRR was possibly related to Xa4 and ZCL-NBS, while not to Xa3. From the expression analysis, several genes of different structure such as Xa26, ZCL-RK and ZCL-NBS located in Xa22(i) region. Xa22(t) might tightly link to alleles ofXa4 andXa26, andXa4 might be the closer one.(4) Evolution of Xa22(t) regionMany genes of different structure tightly linked in Xa22(i) region. Two typical evolutionary model of resistance genes were found in evolution of Xa22(t) region. One model was similar to flax L model, where many alleles of Xa26 were found in IRBB3, Minghui63, Nipponbare and 9311 and they might be orthologs in evolution. Another model was similar to flax M model for many members were adjacent to Xa26 alleles in Minghui63 and Nipponbare. In general, the identity between orhtolo...
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