| Bacterial blight is a common bacterial disease caused by pathogen Xanthomonas oryzae pv.oryzae (Xoo),infecting rice leaves.Breeding new resistant rice cultivars would be an effective,economic and environmental approach to better control the disease.Yunnan Province,known as one of the origin centers of cultivated rice in Asia,is rich in rice landrace resources.It has been an important direction of utilization of rice resources in Yunnan Province to explore new genes resistant to bacterial blight by modern biotechnology.In this study,a total of 200 rice landraces after preliminary screening from 16 states/cities of five rice cultivation areas in Yunnan Province were inoculated with 10 strong pathogenic Xoo strains to conduct accurate evaluation and identification the bacterial blight resistance.We then screened different resistant rice landraces to identify and analyze the distribution characteristics of previously 14 cloned bacterial blight resistance genes.The purpose is to locate new bacterial blight resistance genes by screening resource materials with the possibility of a novel gene resistance to bacterial blight,and provide a foundation for further research and untilization.The main results were as follow:1.Ten Xoo strains including PXO99,T7147,C5,C9,Y8,YM1,YM187,Hzhj19,YJdp2 and YJws2 were inoculated with 200 rice landraces to evaluate the phenotypic resistance to bacterial blight for two consecutive years.The results showed that among the tested local rice resources,the PXO99 strain had the strongest pathogenicity with an average lesion ratio of 0.50,and the YM187 strain has the weakest pathogenicity with an average lesion ratio of 0.27.Meanwhile,the resistance levels and resistance responses of 10 Xoo strains from different rice cultivation areas,states (cities) regions,and indica/japonica subspecies in Yunnan Province were different.Sixty-six local rice landraces were screened for resistance to at least one strain,of which 46 accessions were resistant to one strain,15 accessions were resistant to two strains,two accessions were resistant to three strains,one accession was resistant to six strains (Huangxiangnuo from Honghe State),one accession was resistant to nine strains (Laoyaling from Lincang City) and one accession was resistant to ten strains (Xilandigu from Baoshan City).There were relatively more resistant resources in the Ⅱ and Ⅲ rice cultivation areas (Baoshan and Lincang regions).Comparing and analyzing the resistance spectrum of positive control materials with known resistance genes,it was found that the resistance spectrum of most resistant materials were different from that of the positive control groups,and it was speculated that there may be other or new bacterial blight resistance genes.2.The molecular markers of 14 cloned bacterial blight resistance genes Xa1(Xa2/Xa31(t),Xa14,Xa45(t)),Xa3/Xa26,Xa4,xa5,Xa7,Xa10,xa13,Xa21,Xa23,xa25 and Xa27 were used to detect and analyze homologous resistance alleles in 66 resistant materials.The results showed that two resistance allelic fragments of xa13 and Xa21 were not detected in the 66 resistant materials.The above allelic markers of disease resistance gene were not detected in 3 materials (Lengshuigu from Yuxi City,Laoyaling from Lincang City and Xilandigu from Baoshan City).The frequency of other gene resistance alleles in resistant materials was Xa1(Xa2/Xa31(t),Xa14,Xa45(t)) 56.1% (37),Xa3/Xa26 10.6% (7),Xa4 47.0% (31),xa5 22.7% (15),Xa7 12.1%(8),Xa10 7.6% (5),Xa23 6.1% (4),xa25 40.9% (27) and Xa27 53.0% (35).Most of the materials contained more than 2 molecular marker fragments of the above genes,with a maximum of 5,but did not show broad-spectrum resistance.3.Homologous gene sequence cloning method was used to design gene-specific primers for gene coding region and functional polymorphism region to further check and verify the materials containing the above resistance allele markers.It was found that the above materials containing resistance allelic markers did not contain Xa27 and Xa45(t) homologous genes,but contained Xa1,Xa2/Xa31(t),Xa14,Xa3/Xa26,Xa4,xa5,Xa7,Xa10,Xa23 and xa25 homologous genes,and the corresponding numbers were 9,24,4,7,9,18,1,5,4 and 27,respectively.Meanwhile,3 materials without the above 14 resistance allelic markers were selected as controls to amplify homologous fragments.The results indicated that they contained the same fragments as Xa3/Xa26,xa5 and xa25.Furthermore,nucleotide sequence comparison,protein domain prediction and gene expression analysis were performed on the above resistant materials containing 10 disease-resistant homologous genes,respectively.The results showed that the resistant materials contained the Xa1,Xa2/Xa31(t),Xa14,Xa3/Xa26,Xa5,Xa10,xa23 and xa25Nip homologous genes,but not disease resistance genes due to the variable nucleotide sequences of these resistant materials.And the homologous genes were subjected to different selection pressures in different nucleotide regions,resulting differences in gene expression.Only one accession Qishanggu from Wenshan State and one accession Hongjiugu from Zhaotong City were detected to contain Xa7 and xa25Nip disease resistance genes,respectively.Three resistant materials without the above disease-resistant allelic markers were identify to contain xa3/xa26,Xa5 and Xa25zs97 (Lengshuigu from Yuxi City and Laoyaling from Lincang City)/Xa25IR24 (Xilandigu from Baoshan City) susceptible homologous gene types,indicating these three materials may have potential new genes for resistance to bacterial blight.4.Xilandigu,a broad-spectrum indica rice variety that did not contain the above 14 resistance genes but was resistant to the above 10 strains,was selected as the mapping material of the new gene.Using Xilandigu as the male parent and 02428 as the female parent were constructed F2 population.We obtained 1 276 F2 population individual plants inoculated with PXO99 strain for genetic analysis of resistance.The resistance and susceptibility separation ratio was 3:1 in F2 population,which was believed to contain a dominant resistance gene,temporarily named X63(t) according to material number 63.The X63(t) was located between the markers A11P25204 and RM224 at the end of the long arm of chromosome 11,and the genetic distance was 9.4 cM among the two markers.Compared with the position of the known bacterial blight resistance gene on rice chromosome 11,X63(t) was considered to be a new bacterial blight resistance gene,because its position was different from the known bacterial blight resistance genes. |
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