分子标记辅助聚合3个稻瘟病抗性基因
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摘要
稻瘟病是水稻三大病害之一,我国目前推广的水稻品种抗性基因单一,而且杂交稻几乎不抗稻瘟病,故近年来该病害呈上升趋势。在育种工作中培育抗性品种是最经济有效的方法之一。金23A是一个配合力较好米质较优的不育系,但由于不抗稻瘟病,限制了该不育系的推广应用。本研究以C101LAC(携带Pi-1、Pi-33抗稻瘟病基因)和C101A51(携带Pi-2抗稻瘟病基因)为供体亲本,以金23B为轮回亲本,将分子标记技术与常规育种方法相结合,开展了抗性基因聚合研究。主要
研究结果如下:
1、用分别带有Pi-1、Pi-2和Pi-33的3份材料C104LAC、C101A51和北京糯筛选到特异性鉴定菌株4个:PH14、CH131、CH43和ZJ42。
2、根据已公布的水稻基因组信息,通过筛选分别获得了与抗稻瘟病基因Pi-1、Pi-2、Pi-33紧密连锁的多态性SSR标记:RM224、RM527、RM3215。
3、利用SSR标记RM224对Pi-1选择的准确率在94.4%以上,而用菌株PH14对Pi-1进行选择,则准确率为87.86%,同时对Pi-1进行选择的准确率可达到99.32%。RM527对Pi-2选择的准确率为100%,而用菌株CH131对Pi-1进行选择,则准确率为84.15%,同时利用标记和菌株进行选择的准确率在100%。RM3215对Pi-33选择的准确率为98.73%,菌株CH43对Pi-33选择的准确率为88.14%,同时选择的理论准确率在99.85%以上。
4、F1代,结合与上述3个基因紧密连锁的分子标记的检测结果和特异性鉴别菌株的鉴定结果,选到了聚合3个抗性基因、且基因型杂合的单株35株;选择其中性状优异的4个单株种植成400个单株的F2的群体,通过标记检测和菌株验证获得了聚合3个抗性基因、目标位点基因型纯合的单株5株。
5、利用30个菌株对选择聚合3基因的过程中得到的抗性基因两两聚合的单株W1、W2、W3,和聚合3个基因的单株W4,进行抗谱分析。结果是:2个基因的聚合系对稻瘟病的抗性频谱达到73.3～86.7%,抗谱均明显宽于单个基因Pi-1、Pi-2和Pi-33的C104LAC、C101A51和北京糯;而聚合Pi-1、Pi-2和Pi-33抗性基因的W4仅仅能被1个菌株侵染,抗病谱频率达到96.7%。
Rice blast, caused by Magnaporthe grisea, is the most serious disease in rice (Oryza sativa L). Most of rice varieties planted in China only carried a little number of resistance genes and were susceptible to blast. Consequently, the damage by blast in rice presented an increasing trend in recent years. Developing resistant varieties to blast is one of the most economical and effective methods. Jin 23B is an excellent CMS maintain-line with good qualities and combining ability except for blast resistance. This study was mainly to pyramid three blast resistant genes into Jin 23B by marker-assisted selection.
The donors, carried different resistance genes, were C101LAC (carried Pi-1 and Pi-33 resistance genes) and C101A51 (carried Pi-2 gene), respectively. In process of pyramiding Pi-1, Pi-2 and Pi-33, two methods, inoculation identification and marker assisted selection, were carried out. The main results were as follows:
1) Four specific rice blast isolations (PH14, CH131, CH43 and ZJ42), were screened out on the base of identification results, Of which PH14 and CH43 could not infect C104LAC (carried Pi-1) and Bei-jing-nuo (carried Pi-33) respectively, while two isolations, CH131 and ZJ42, could not infect C101A51 (carried Pi-2). These selected isolations were used to inoculation identification for distinguishing three resistance genes, Pi-1, Pi-2 and Pi-33.
2) Based on the markers information of rice and the fine mapping results of resistant genes, Pi-1, Pi-2 and Pi-33, three SSR markers (RM224, RM527 and RM3215) flanked to these genes, were used in the process of marker-assisted selection (MAS), respectively.
3) The correct ration was 94.4% with molecular marker RM224 for selecting Pi-1 gene on segregating generation, while the correct ration was only 87.86% when PH14 isolation was used to select this gene. However, if combined the two methods, RM224 and PH14, the correct ration could up to 99.32%. The similar results were found in the other two genes, Pi-2 and Pi-33, which indicated the method of molecular marker was better than that of inoculation identification in selecting target gene.
4) Applying both SSR markers and specific isolates in multiple F1 generation, the 35 plants were acquired with heterozygous genotype of 3 blast resistant genes, and four of them with much more similar to Jin 23 were selected. In progeny of the selected four plants, five plants with homozygous genotype of Pi-1, Pi-2 and Pi-33 were selected.
5) With 30 isolates, we identified four lines, W1 carried Pi-1 and Pi-2, W2 carried Pi-2 and Pi-33, W3 carried Pi-1 and Pi-33, and W4 pyramiding 3 genes Pi-1, Pi-2 and Pi-33. The results indicated that the spectrum of lines pyramiding 2 blast resistant genes ranged 73.3% to 86.7%, which was broader than that of lines carried only one gene, C104LAC, C101A51 and Bei-jing-nuo. However, the spectrum of W4 was close to 100% (96.7%) for pyramiding 3 genes, and just one isolate used in this study could infect this line.
研究结果如下:
1、用分别带有Pi-1、Pi-2和Pi-33的3份材料C104LAC、C101A51和北京糯筛选到特异性鉴定菌株4个:PH14、CH131、CH43和ZJ42。
2、根据已公布的水稻基因组信息,通过筛选分别获得了与抗稻瘟病基因Pi-1、Pi-2、Pi-33紧密连锁的多态性SSR标记:RM224、RM527、RM3215。
3、利用SSR标记RM224对Pi-1选择的准确率在94.4%以上,而用菌株PH14对Pi-1进行选择,则准确率为87.86%,同时对Pi-1进行选择的准确率可达到99.32%。RM527对Pi-2选择的准确率为100%,而用菌株CH131对Pi-1进行选择,则准确率为84.15%,同时利用标记和菌株进行选择的准确率在100%。RM3215对Pi-33选择的准确率为98.73%,菌株CH43对Pi-33选择的准确率为88.14%,同时选择的理论准确率在99.85%以上。
4、F1代,结合与上述3个基因紧密连锁的分子标记的检测结果和特异性鉴别菌株的鉴定结果,选到了聚合3个抗性基因、且基因型杂合的单株35株;选择其中性状优异的4个单株种植成400个单株的F2的群体,通过标记检测和菌株验证获得了聚合3个抗性基因、目标位点基因型纯合的单株5株。
5、利用30个菌株对选择聚合3基因的过程中得到的抗性基因两两聚合的单株W1、W2、W3,和聚合3个基因的单株W4,进行抗谱分析。结果是:2个基因的聚合系对稻瘟病的抗性频谱达到73.3～86.7%,抗谱均明显宽于单个基因Pi-1、Pi-2和Pi-33的C104LAC、C101A51和北京糯;而聚合Pi-1、Pi-2和Pi-33抗性基因的W4仅仅能被1个菌株侵染,抗病谱频率达到96.7%。
Rice blast, caused by Magnaporthe grisea, is the most serious disease in rice (Oryza sativa L). Most of rice varieties planted in China only carried a little number of resistance genes and were susceptible to blast. Consequently, the damage by blast in rice presented an increasing trend in recent years. Developing resistant varieties to blast is one of the most economical and effective methods. Jin 23B is an excellent CMS maintain-line with good qualities and combining ability except for blast resistance. This study was mainly to pyramid three blast resistant genes into Jin 23B by marker-assisted selection.
The donors, carried different resistance genes, were C101LAC (carried Pi-1 and Pi-33 resistance genes) and C101A51 (carried Pi-2 gene), respectively. In process of pyramiding Pi-1, Pi-2 and Pi-33, two methods, inoculation identification and marker assisted selection, were carried out. The main results were as follows:
1) Four specific rice blast isolations (PH14, CH131, CH43 and ZJ42), were screened out on the base of identification results, Of which PH14 and CH43 could not infect C104LAC (carried Pi-1) and Bei-jing-nuo (carried Pi-33) respectively, while two isolations, CH131 and ZJ42, could not infect C101A51 (carried Pi-2). These selected isolations were used to inoculation identification for distinguishing three resistance genes, Pi-1, Pi-2 and Pi-33.
2) Based on the markers information of rice and the fine mapping results of resistant genes, Pi-1, Pi-2 and Pi-33, three SSR markers (RM224, RM527 and RM3215) flanked to these genes, were used in the process of marker-assisted selection (MAS), respectively.
3) The correct ration was 94.4% with molecular marker RM224 for selecting Pi-1 gene on segregating generation, while the correct ration was only 87.86% when PH14 isolation was used to select this gene. However, if combined the two methods, RM224 and PH14, the correct ration could up to 99.32%. The similar results were found in the other two genes, Pi-2 and Pi-33, which indicated the method of molecular marker was better than that of inoculation identification in selecting target gene.
4) Applying both SSR markers and specific isolates in multiple F1 generation, the 35 plants were acquired with heterozygous genotype of 3 blast resistant genes, and four of them with much more similar to Jin 23 were selected. In progeny of the selected four plants, five plants with homozygous genotype of Pi-1, Pi-2 and Pi-33 were selected.
5) With 30 isolates, we identified four lines, W1 carried Pi-1 and Pi-2, W2 carried Pi-2 and Pi-33, W3 carried Pi-1 and Pi-33, and W4 pyramiding 3 genes Pi-1, Pi-2 and Pi-33. The results indicated that the spectrum of lines pyramiding 2 blast resistant genes ranged 73.3% to 86.7%, which was broader than that of lines carried only one gene, C104LAC, C101A51 and Bei-jing-nuo. However, the spectrum of W4 was close to 100% (96.7%) for pyramiding 3 genes, and just one isolate used in this study could infect this line.
引文
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99、Yu ZH,Mackill DJ,Bonman JM,et al.,Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet,1991,81:471~476。
100、Yu ZH,Mackill DJ,Borunan JM,Molecular mapping of genes for resistance to rice blast (Pyricularja grisea Sacc.),Theor. Appl. Genet,1996,93:859~863。
101、Zhu X,Shen Y,Adreit H et al,Resistance evaluation of some Chinese leading rice maintainer,restorer lines and their hybrids to Magnaporthe grisea,Rice Science, 2004,11(3):101~105。
102、D.J. Mackill, J.M. Bonman et al. Near-isogenic Indica rice lines with blast resistance genes,Rice Genetics Newsletter, Vol. 5:98 ~ 100。