水稻品种抗瘟遗传多样性及抗瘟基因分子标记研究
|
摘要
用抗病基因保守区域合成的38个引物组合成28个引物对，对中国水稻丽江新团黑谷(LTH)近等基因系品种进行扩增。结果表明，不同引物对对同一近等基因系的扩增图谱有很大差异。引物对LM637／LM638扩增谱带最多，共40条；Ptokin1N／Ptokin2N仅扩增1～2条模糊的条带；而Ptokin2-inv1／Ptokin2-inv2扩增后无条带出现。此外，同一引物对(S2／AS1)对不同近等基因系(LTH与Co39)扩增的图谱亦有较大差异。
选择扩增谱带丰富且据不同类型保守区域设计的3对引物XLRRfor／XLRRrev、S1／AS3和Ptokin1／Ptokin2，对江苏27个品种进行扩增。结果指出，3对引物共扩增出清晰可辨的谱带127条，其中多态性带101条，占总数的79.53％。3对引物中扩增谱带和多态性带频率最高的是XLRRfor／XLRRrev，共扩增谱带47条，其中多态性带40条，占85.11％。此外，3对引物扩增的多态性谱带的分子量大小基本无差异。
根据聚类分析，3对引物对或引物组合扩增的RGA图谱可将品种分为多种类型，这些RGA图谱类型与病菌7个小种接种鉴定的抗病表型没有完全的对应关系。其中XLRRfor／XLRRrev引物对其组合的RGA图谱类型似乎与抗病表型有较好的吻合程度，这可能是由于该引物对来自水稻之缘故。
以28对引物对LTH近等基因系品种进行扩增，有11对引物能扩增出特异性条带，共回收特异性片段33个。将回收的片段重新扩增，有11个片段获单一条带。选择其中两个480～490bp片段(HS-1和HS-19)进行探针标记。点杂交试验表明，所有供试品种都有杂交信号，这说明这些片段在各品种中都有相似的序列或部分同源序列。PCR扩增产物电泳后经Southern杂交发现，片段HS-1在含有Pi-ta~2抗性基因的品种F-128-1、NO4中有特异的杂交带出现，这表明此序列可能与抗性基因Pi-ta~2连锁或是抗性基因的一部分。
用引物对XLRRfor／XLRRrev对202个品种进行扩增，在480bp处出现特异带
扬州大学硕士学位论文
的品种有34个.但与抗性鉴定结果比较发现，即使具有相同特异带的品种其抗病
表型也是多样的，这可能与品种抗病表型的遗传复杂性有关。
将特异性片段HS一l克隆测序，全长为478bp.通过在Genbank中进行检索，
发现它与Mag。等(1999)在水稻中克隆的一个抗病基因同源序列RGA29有95%
的同源性。
38 primers synthesized according to resistance gene conserved domains were made up 28 primer pairs. These primer pairs were used to amplify LTH NILs in China. The results showed that the amplified band types in same NILs varied with primer pairs used. The primer pair LM637/LM638 could amplify 40 bands from the varieties, which was the most among 3 primer pairs. Only 1 to 2 bands were amplified by Ptokin1N/Ptokin2N while no band appeared in the amplification of Ptokin2-invl/Ptokin2-inv2. In addition, the bands amplified by the primer pair S1/AS3 were significantly different in two NILs (LTH and Co39).
3 primer pairs including XLRRfor/XLRRrev, Ptokin1/Ptokin2 and S1/AS3 were selected to amplified 27 rice cultivars from Jiangsu Province. These primer pairs were designed according to different resistance gene conserved domains and had high ability of the amplification. 127 bands were amplified from 27 rice cultivars by 3 primer pairs, in which 101 bands were polymorphic, with the polymorphic band frequency of 79.53%. The frequency of polymorphic bands amplified by XLRRfor/XLRRrev was the highest among 3 primer pairs, with the sum of 47 bands and the polymorphic band frequency of 85.11%. Furthermore, there were no differences in the molecular weight of polymorphic bands amplified by 3 primer pairs.
RGA-PCR maps of rice cultivars amplified by 3 primer pairs or primer pair combinations could be grouped by cluster analysis. There were no completely corresponding relations between these RGA-PCR map groups and resistance phenotypes of rice cultivars. But RGA-PCR maps amplified by XLRRfor/XLRRrev or its combinations had same relations with the resistance phenotypes, which might be because the primer pair came from rice.
28 primer pairs were used to amplify LTH NILs varieties. There were 11 primer pairs by which special bands could be amplified. 33 special bands were retrieved and re-amplified, and 11 simple bands were acquired. Two fragments (HS-1 and HS-19) with the size from 480bp to 490bp were selected to sign as probes. There were same
signals in all rice varieties by dot hybridization, which indicated that all rice varieties tested could have same or partial RGA.
The results of electrophoresis and southern hybridization showed that HS-1 fragment could detect special signals in rice varieties with resistance gene Pi-ta2, which revealed that this sequence could link with Pi-ta2 gene or be a part of the gene.
34 rice varieties had the special band of 480bp among 202 rice varieties amplified with XLRRfor/XLRRrev. By comparison, it was found that although rice varieties had the same special band, their resistance phenotypes could be different, which maybe related to genetic complexity of resistance phenotypes.
HS-1 fragment has been cloned and sequenced, and was 478bp. It was 95% identical with RGA29 which was a resistance gene analog cloned from rice by Mago(1999).
选择扩增谱带丰富且据不同类型保守区域设计的3对引物XLRRfor／XLRRrev、S1／AS3和Ptokin1／Ptokin2，对江苏27个品种进行扩增。结果指出，3对引物共扩增出清晰可辨的谱带127条，其中多态性带101条，占总数的79.53％。3对引物中扩增谱带和多态性带频率最高的是XLRRfor／XLRRrev，共扩增谱带47条，其中多态性带40条，占85.11％。此外，3对引物扩增的多态性谱带的分子量大小基本无差异。
根据聚类分析，3对引物对或引物组合扩增的RGA图谱可将品种分为多种类型，这些RGA图谱类型与病菌7个小种接种鉴定的抗病表型没有完全的对应关系。其中XLRRfor／XLRRrev引物对其组合的RGA图谱类型似乎与抗病表型有较好的吻合程度，这可能是由于该引物对来自水稻之缘故。
以28对引物对LTH近等基因系品种进行扩增，有11对引物能扩增出特异性条带，共回收特异性片段33个。将回收的片段重新扩增，有11个片段获单一条带。选择其中两个480～490bp片段(HS-1和HS-19)进行探针标记。点杂交试验表明，所有供试品种都有杂交信号，这说明这些片段在各品种中都有相似的序列或部分同源序列。PCR扩增产物电泳后经Southern杂交发现，片段HS-1在含有Pi-ta~2抗性基因的品种F-128-1、NO4中有特异的杂交带出现，这表明此序列可能与抗性基因Pi-ta~2连锁或是抗性基因的一部分。
用引物对XLRRfor／XLRRrev对202个品种进行扩增，在480bp处出现特异带
扬州大学硕士学位论文
的品种有34个.但与抗性鉴定结果比较发现，即使具有相同特异带的品种其抗病
表型也是多样的，这可能与品种抗病表型的遗传复杂性有关。
将特异性片段HS一l克隆测序，全长为478bp.通过在Genbank中进行检索，
发现它与Mag。等(1999)在水稻中克隆的一个抗病基因同源序列RGA29有95%
的同源性。
38 primers synthesized according to resistance gene conserved domains were made up 28 primer pairs. These primer pairs were used to amplify LTH NILs in China. The results showed that the amplified band types in same NILs varied with primer pairs used. The primer pair LM637/LM638 could amplify 40 bands from the varieties, which was the most among 3 primer pairs. Only 1 to 2 bands were amplified by Ptokin1N/Ptokin2N while no band appeared in the amplification of Ptokin2-invl/Ptokin2-inv2. In addition, the bands amplified by the primer pair S1/AS3 were significantly different in two NILs (LTH and Co39).
3 primer pairs including XLRRfor/XLRRrev, Ptokin1/Ptokin2 and S1/AS3 were selected to amplified 27 rice cultivars from Jiangsu Province. These primer pairs were designed according to different resistance gene conserved domains and had high ability of the amplification. 127 bands were amplified from 27 rice cultivars by 3 primer pairs, in which 101 bands were polymorphic, with the polymorphic band frequency of 79.53%. The frequency of polymorphic bands amplified by XLRRfor/XLRRrev was the highest among 3 primer pairs, with the sum of 47 bands and the polymorphic band frequency of 85.11%. Furthermore, there were no differences in the molecular weight of polymorphic bands amplified by 3 primer pairs.
RGA-PCR maps of rice cultivars amplified by 3 primer pairs or primer pair combinations could be grouped by cluster analysis. There were no completely corresponding relations between these RGA-PCR map groups and resistance phenotypes of rice cultivars. But RGA-PCR maps amplified by XLRRfor/XLRRrev or its combinations had same relations with the resistance phenotypes, which might be because the primer pair came from rice.
28 primer pairs were used to amplify LTH NILs varieties. There were 11 primer pairs by which special bands could be amplified. 33 special bands were retrieved and re-amplified, and 11 simple bands were acquired. Two fragments (HS-1 and HS-19) with the size from 480bp to 490bp were selected to sign as probes. There were same
signals in all rice varieties by dot hybridization, which indicated that all rice varieties tested could have same or partial RGA.
The results of electrophoresis and southern hybridization showed that HS-1 fragment could detect special signals in rice varieties with resistance gene Pi-ta2, which revealed that this sequence could link with Pi-ta2 gene or be a part of the gene.
34 rice varieties had the special band of 480bp among 202 rice varieties amplified with XLRRfor/XLRRrev. By comparison, it was found that although rice varieties had the same special band, their resistance phenotypes could be different, which maybe related to genetic complexity of resistance phenotypes.
HS-1 fragment has been cloned and sequenced, and was 478bp. It was 95% identical with RGA29 which was a resistance gene analog cloned from rice by Mago(1999).
引文
1 Ou, S. H.. Rice disease. Farnham Royal, UK: Commonwealth Agricultural Bureaux, 1985.
2 Baker, B., Zamryski, P., Staskawicz, B., et al. Signaling in plant microbe interactions. Science, 1997, 276: 726～733.
3 董继新，董海涛，李德葆．水稻抗瘟性研究进展．农业生物技术学报，2000，8(1)：99～102．
4 朱祯，刘翔．转基因作物—恶魔还是救星．农业生物技术学报，2000，8(1)：1～5．
5 孙漱沅，孙国昌．我国稻瘟病研究的现状和展望．植保技术与推广，1996，(3)：39～40．
6 山崎義人，高坂卓爾编著(1980，凌忠专、孙昌其译)．稻瘟病与抗病育种．农业出版社，1990，pp．19～31，88～134，170～224，352～482，507～550．
7 熊振民，蔡洪法，闵绍楷等．中国水稻．中国农业科技出版社，1992，20～35．
8 彭绍裘，刘二明，黄费元等．水稻持久抗瘟性研究．植物保护学报，1996，23(4)：293～299
9 Ahn, S.W. Identification of a fourth blast resistance gene in the Vietnamese indica cultivar Tetep. Rice Genetics Newsletter, 1994, 12: 237～238.
10 Kiyosawa, S. Establishment of differential varieties for pathogenicity test of rice blast fungus. Rice Genetics Newsletters, 1994, Volumn (1): 95～96.
11 Yu, Z. H., Mackill, D. J., Bonman, J. M. et al. Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet.1991, 81:471～476.
12 Yu, Y. G., Buss, G. R., Saghai-Maroof, M. A. Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site. Proc Natl Acad Sci USA, 1996, 93: 11751～11756.
13 Wang, G.L., Mackill, D. J., Bonman, J. M., et al. RFLP mapping of genes conferring complete and partial resistance of blast in a durably resistant rice cultivar. Genetics, 1994, 136:1421～1434.
14 Zheng, K., Subudhi, P.K., Domingo, J., et al. Rapid DNA isolation for marker assisted selection in rice breeding. Rice Genetics Newsletter. 1995,12:255～258.
15 Zhu, L. H., Y. Chen, Y. B., Xu, J. C. et al. Construction of a molecular map of rice and gene mapping using a double haploid population of cross between Indica and Japonica varieties. RGN, 1993, 10:132～135.
16 MeCouch, S.R., Nelson, R.J., Tohme, J., et al. Mapping of blast resistance genes in rice .In Zeiger RS,Leong SA,Teng PS(eds):Rice blast disease, CAB International and IRRI Press, 1994,
167～186.
17 Zhuang, J. Y., J. L. Wu, R.Y. Chai, et al. Rapid identification of resistance gene analogs linked to Iwo blast resistance genes in rice. RGN, 1998, 15: 125～127.
18 Chen, X. M., Line, R. F. Leung, H. et al. Genome scanning for resistance gene analogs in rice, barley and wheat by high resolution electrophoresis. Theor. Appl. Genet., 1998, 97:345～355.
19 李月莹，裴忠有，沈平等．分子标记技术及其在作物遗传育种研究上的应用．沈阳学业大学学报，2001，32(2)：150～154．
20 黄大年，杨炜，王金霞等．利用遗传同质的近等基因对研究水稻抗稻瘟病基因的产物．中国水稻科学，1992，6(2)：49～52．
21 Peng, Y. L., Shirano, Y., Ohta, H. et al. A novel lipoxygenase from rice,Primary structure and specific expression upon incompatible infection with rice blast fungus. Biol. Chem. 1994, 269: 3755～3761.
22 Chen, B. Valent, R. J. Nelson. The relationship between lineage and virulence in Pyricularia grisea in the Philippines. Phytopathology, 1995, 85:443～451.
23 Mauch, F., Reimmann, C., Freydl, E., et al. Characterization of the rice pathogen-related protein Rirlaand regulation of the corresponding gene. Plant Mol. Biol., 1998, 38, 577～586.
24 董海涛，何祖华，吴玉良．水稻抗稻瘟病近等基因系mRNA差别显示分析．农业生物技术学报，1998，6(3)：223～227．
25 Jensen, N. F. Intra-varietal diversification in oat breeding. Agronomy Journal, 1952, 44: 30～44.
26 Priestly, J. H. Choise and development of resistant cultivars for cereal diseases control. In: Strategies for the Control of Cereal disease. Jenkyn, J. F. and R. T. Plumb (ed.). Blackwel, Oxford, 1981, 65～72.
27 Vanderplank, J. E. Disease Resistance in Plants. Academ. Press, New York. 1968.
28 Knott, E.A., Mundt, C.C. Mixing ability analysis of wheat cultivar mixtures under diseased and nondiseased conditions. Theor. Appl. Genet. 1990, 80:313～320.
29 Reddy, M. S., Rao, M.V. Resistance genes and their deployment for control of leaf rust of wheat. Indian J. Genet. Plant Breed. 1979,39: 359～365.
30 Mundt, C.C., Brophy, L. S. Influence of number of host genotype units on the effectiveness of host mixtures for disease control: a modeling approach. Phytopath. 1988, 75:1141～1145.
31 Browning, J.A., Simmons, M. D., Frey, K.J. Regional deployment for conservation of oat crown rust resistance genes. In: Disease Consequences of Intensive and Extensive Culture of Field rops.Browning, J. A. (ed.). Iowa Agriculture and Home Economics Special Report 64. Iowa State University Press, Ames, Iowa, 1969, 49～56.
32 吕亮，陈其志，陈茂华等．不同水稻品种间栽控制稻瘟病的田间试验．华中农业大学学报2002，21(3)：228～230．
33 Frey, K. J., Browning, J. A. Simons M D. Management systems for host genes to control disease loss. Ann. N. Y. Acad. Sci. 1977, 255～287.
34 Wolfe, M. S. Barley diseases: maintaining the value of our varieties. In: Barley Genetics Ⅵ, vol. 2. Munck, L. (ed.). Munksgaard International Publishers, Copenhagen, Denmark, 1992, 1055～1067.
35 毛建辉，何明，何忠全．抗感品种混植对水稻主要病害的效应．植物病理学报，1991，21(2)：155～160．
36 Flor, H. Current status of the gene-for-gene concept. Annu Rev Phytopathol, 1997, 9: 275～296.
37 张冬生．《植物体细胞遗传学》，1987，复旦大学出版社．
38 刘植义，刘彭昌，周希澄等，《遗传学》，1982，人民教育出版社．
39 龙跃生，陈良碧，夏快飞等．分子标记技术及其在分离和克隆水稻基因中的应用．生物学杂志，2001，18(2)：29～32．
40 李柱刚，崔崇士，马荣才等．遗传标记在植物上的发展与应用．东北农业大学学报，2001，33(4)：313～319．
41 王和勇，陈敏，廖志化等．RFLP、RAPD、AFLP分子标记及其在植物生物技术中的应用，生物学杂志，1998，15(4)：24～26．
42 Bststein, D., White, R.L., Skolnick, M., et al. Construction of a genetic map in man use restiction fragment polymorphisms. Genet, 1980, (32): 314～3311.
43 孙雁，王云月，何月秋等．水稻抗病基因同源序列多态性与品种鉴定．种子，2001，(2)：1～2．
44 Wang, G.L., Mackill, D.J., Bonman, J.M., et al.. RFLP mapping of genes conferring complete and partial resistance to blast in a durably resistant rice cultivar. Genetics, 1994, 136: 1421～1434.
45 章琦，赵炳宇，赵开军等．普通野生稻的抗水稻白叶枯病(Xanthomonas oryzae pv.oryzae)新基因Xa-23的鉴定和分子标记．作物学报，2000，26(5)：536～542．
46 吴建利，庄杰云，柴荣耀等．水稻抗穗瘟基因的分子定位．植物病理学报，2000，30(2)：111～115．
47 聂练兵．转基因作物及其检测技术与安全性评价问题．种子科技．2001，(3)：152～154．
48 李常保．RAPD标记与作物改良．生物技术通报．1998，6：20～29．
49 赵建伟，孟金陵．植物抗病基因的克隆及其在作物遗传改良中的意义．高技术通讯，1998，58～62．
50 Wiliams, J.C.K., Kubelik, A.R.J., Livak, K,, et al.. DNA polymorphism amplified by arbitrary primes are useful as genetic markers. Nucleic Acide Reseach, 1990, 18(22):6531～6535.
51 Leister, D., Ballvora, A., Salamini, F., et al. Nature Genetics, 1996, 14: 421～429.
52 Kanazin, V., Marek, L. F., Shoemaker, R. C. Resistance gene analogs are conserved and clustered in soyabean. Proc Natl. Acad. Sci. USA, 1996, 93: 11746～11750.
53 Yu, Y. G., Buss, G. L., Maroof, M. A. S. Isolation of a super-family of candidate disease resistance genes in soyabean based on a conserved nucleotide-binding site. Proc Natl. Acad. Sci. USA, 1996, 93: 11751～11756.
54 Brune,I D., Froger, N., Pelletier, G. Development of amplified consensus genetic marker (ACGM) in Brassica napus from Arabidopsis thaliana sequences of known biological function, Genome. 1999, (42): 387-402.
55 Yu. Z. H., D. J. Mackill, J. M. Bonman, et al. Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet. 1991, 81(4): 471～476.
56 Yu, Z.H., Mackill, D.J., Bonman, J.M., et al. Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.). Theor. Appl. Genet. 1996, 93, 859～863.
57 Li-Huang Zhu, Ying Chen, Yun-Bin Xu, et al. Construction of a molecular map of rice and gene mapping using a double haploid population of a cross between Indica and Japonica varieties, Rice Genetics Newsletter, 1999, (10) 132～134.
58 Wang D.L., J. Zhu, Z.K. Li, et al.. Mapping QTLs with epistatic effects and QTLx environment interactions by mixed linear model approaches. Theor. Appl. Genet. 1999, 99:1256～1264.
59 Inukai, T., R. J. Nelson, R. S. Zeigler, et al. Allelism of blast resistance genes in near-isogenic lines of rice. Phytopathology, 1994, 84: 1278～1283.
60 T. Inukai, D. J. Mackill, P. C. Ronald, et al. Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar. Theor Appl Genet, 1999, 98: 1046～1053.
61 Wang, Z. X., Yano, M., Yamanouchi, et al. The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine- rich repeat class of plant disease esistance genes. Plant. 1999, 19, 55～64.
62 Mindrinos, M., Katagiri, F., Yu, G. L., et al. The Arabidopsis thaliana disease resistance gene RPS2 encodes a pro-tein containing a nucleotide- binding site and leucine- rich repeat. Cell, 1994, 78: 1089-1099.
63 Grant, M. R., Godiard, L., Straube, E., et al. Structure of the Arabidopsis RPM1 gene enable dual specificity di-sease resistance. Science, 1995, 269: 843～846.
64 Jones, D. A., Thomas, C. M.,Hammond- kosack, K. E., et al. Isolation of the tomato Cf- 9gene for resistance to Cladosporium fulvum by transposon tagging. Science, 1994, 266: 789～793.
65 Thomas, C. M., Jonesd, A., Parniske, M., et al. Characterization of the tomato Cf- 4gene for resistance to Cladosporium fulvum identifies sequences thatdetermine recognitional specificity in Cf- 4 and Cf- 9. Plant Cell, 1997, 9: 2209～2224.
66 Whitham, S., Dinesh- kumar, S.P., Choid, et al. The productof the tobacco mosaic virus resistance gene N: simi-larity to toll and the interieukin-1 receptor. Cell, 1994, 78:1101～1115.
67 Lawrence, G. J., Finnegen, E. J., Ayliffe, M. A., et al. The L6gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N. Plant Cell, 1995, 7:1195～1206.
68 Song, W. Y., Wang, G. L., Chen, L. L., et al. A receptor kinase- like protein encoded by the rice disease resistance gene, Xa2 1. Science, 1995, 270: 1804～1806.
69 Johal, G. S., Briggs, S. P.. Reductase activity encoded by the H m1 disease resistance gene in maize. Science, 1992,258: 985～987.
70 Cai, D., Kleine, M., Kifle, S., et al. Positional cloning of a gene for nematode resistance in sugar beet. Science, 1997, 275: 832～834.
71 李子银，陈受宜．水稻抗病基因同源序列的克隆、定位及表达．科学通报，1999，44(7)：727～733．
72 郑先武，翟文学，李晓兵等．水稻NBS-LRR类R基因同源序列．中国科学，2001，31(1)：43～51．
73 秦跟基，李万隆，陈佩度．植物抗病基因结构特征及其类似序列的研究进展．南京农业大学学报，1999，22(3)：102～107．
74 周玮斌，史晰，詹树萱等．普通野生稻中NBS同源序列的克隆和分析．复旦学报，2001，40
(5)：516～520．
75 易永图．植物抗病基因同源序列及其在抗病基因克隆与定位中的应用．生物技术通报，2002，(2)：16～20．
76 张增艳，孔凡晶，辛志勇．植物抗病基因克隆进展．生物技术通报．2000(2)：8～11
77 周益军．稻瘟病菌遗传多样性和致病性诱变研究．博士论文．2000
78 Pan, Q., wang, L., Tanisaka, T., and Ikehashi. Allelism of rice blast resistance genes in two Chinese rice cultivars, and identification of two new resistance genes. Plant Pathology, 1998, 47, 165～170.
79 McCouch, S. R., Nelson, R. J., Tohem, et al. Mapping of blast resistance genes in rice. In: Rice Blast Disease. CAB INTERNAL, Wallingford, 1994, pp. 167～182.
80 刘二明．水稻品种与稻瘟病菌的遗传多样性及稻瘟病持续控制研究．博士论文，1999．
81 张峰，宋文芹，李凌等．利用AFLP-银染法筛选与抗甘蓝黑腐病性状连锁的分子标记．南开大学学报，1999，32(3)：177～181．
82 刘二明，朱有勇，刘新民等．丘陵区水稻品种多样性混合间栽控制稻瘟病研究．作物研究，2002，16(1)：7～101．
83 王石平，刘克德，王江等．用同源序列的染色体定位寻找水稻抗病基因DNA片段．植物学报，1998，40(1)：42～50．
84 凌忠专，T．Mew，王久林等 中国水稻近等基因系的育成及其稻瘟病菌生理小种鉴别能力．中国农业科学，2000，33(4)：1～8．
85 Callard, D., Axelos, M., Mazzolini, L.. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol. 1996, 112: 705～715..
86 Li, .F, E.S. Barnathan, K. Kariko. Rapid method for screening and cloning cDNA generated in differential display: amplification of Northern blot for affinity capturing of cDNAs. Nucleic Acid Research,1994, 22:1764～1765.
87 孔维文．水稻受白叶枯病菌侵染诱导的抗病相关基因的克隆与鉴定．博士论文，2001
88 Mago, R., Nair, S. and Mohan,M. Resistance gene analogues from rice: cloning, sequencing and mapping. Theor. Appl. Genet. 1999,99: 50～57.
2 Baker, B., Zamryski, P., Staskawicz, B., et al. Signaling in plant microbe interactions. Science, 1997, 276: 726～733.
3 董继新，董海涛，李德葆．水稻抗瘟性研究进展．农业生物技术学报，2000，8(1)：99～102．
4 朱祯，刘翔．转基因作物—恶魔还是救星．农业生物技术学报，2000，8(1)：1～5．
5 孙漱沅，孙国昌．我国稻瘟病研究的现状和展望．植保技术与推广，1996，(3)：39～40．
6 山崎義人，高坂卓爾编著(1980，凌忠专、孙昌其译)．稻瘟病与抗病育种．农业出版社，1990，pp．19～31，88～134，170～224，352～482，507～550．
7 熊振民，蔡洪法，闵绍楷等．中国水稻．中国农业科技出版社，1992，20～35．
8 彭绍裘，刘二明，黄费元等．水稻持久抗瘟性研究．植物保护学报，1996，23(4)：293～299
9 Ahn, S.W. Identification of a fourth blast resistance gene in the Vietnamese indica cultivar Tetep. Rice Genetics Newsletter, 1994, 12: 237～238.
10 Kiyosawa, S. Establishment of differential varieties for pathogenicity test of rice blast fungus. Rice Genetics Newsletters, 1994, Volumn (1): 95～96.
11 Yu, Z. H., Mackill, D. J., Bonman, J. M. et al. Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet.1991, 81:471～476.
12 Yu, Y. G., Buss, G. R., Saghai-Maroof, M. A. Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site. Proc Natl Acad Sci USA, 1996, 93: 11751～11756.
13 Wang, G.L., Mackill, D. J., Bonman, J. M., et al. RFLP mapping of genes conferring complete and partial resistance of blast in a durably resistant rice cultivar. Genetics, 1994, 136:1421～1434.
14 Zheng, K., Subudhi, P.K., Domingo, J., et al. Rapid DNA isolation for marker assisted selection in rice breeding. Rice Genetics Newsletter. 1995,12:255～258.
15 Zhu, L. H., Y. Chen, Y. B., Xu, J. C. et al. Construction of a molecular map of rice and gene mapping using a double haploid population of cross between Indica and Japonica varieties. RGN, 1993, 10:132～135.
16 MeCouch, S.R., Nelson, R.J., Tohme, J., et al. Mapping of blast resistance genes in rice .In Zeiger RS,Leong SA,Teng PS(eds):Rice blast disease, CAB International and IRRI Press, 1994,
167～186.
17 Zhuang, J. Y., J. L. Wu, R.Y. Chai, et al. Rapid identification of resistance gene analogs linked to Iwo blast resistance genes in rice. RGN, 1998, 15: 125～127.
18 Chen, X. M., Line, R. F. Leung, H. et al. Genome scanning for resistance gene analogs in rice, barley and wheat by high resolution electrophoresis. Theor. Appl. Genet., 1998, 97:345～355.
19 李月莹，裴忠有，沈平等．分子标记技术及其在作物遗传育种研究上的应用．沈阳学业大学学报，2001，32(2)：150～154．
20 黄大年，杨炜，王金霞等．利用遗传同质的近等基因对研究水稻抗稻瘟病基因的产物．中国水稻科学，1992，6(2)：49～52．
21 Peng, Y. L., Shirano, Y., Ohta, H. et al. A novel lipoxygenase from rice,Primary structure and specific expression upon incompatible infection with rice blast fungus. Biol. Chem. 1994, 269: 3755～3761.
22 Chen, B. Valent, R. J. Nelson. The relationship between lineage and virulence in Pyricularia grisea in the Philippines. Phytopathology, 1995, 85:443～451.
23 Mauch, F., Reimmann, C., Freydl, E., et al. Characterization of the rice pathogen-related protein Rirlaand regulation of the corresponding gene. Plant Mol. Biol., 1998, 38, 577～586.
24 董海涛，何祖华，吴玉良．水稻抗稻瘟病近等基因系mRNA差别显示分析．农业生物技术学报，1998，6(3)：223～227．
25 Jensen, N. F. Intra-varietal diversification in oat breeding. Agronomy Journal, 1952, 44: 30～44.
26 Priestly, J. H. Choise and development of resistant cultivars for cereal diseases control. In: Strategies for the Control of Cereal disease. Jenkyn, J. F. and R. T. Plumb (ed.). Blackwel, Oxford, 1981, 65～72.
27 Vanderplank, J. E. Disease Resistance in Plants. Academ. Press, New York. 1968.
28 Knott, E.A., Mundt, C.C. Mixing ability analysis of wheat cultivar mixtures under diseased and nondiseased conditions. Theor. Appl. Genet. 1990, 80:313～320.
29 Reddy, M. S., Rao, M.V. Resistance genes and their deployment for control of leaf rust of wheat. Indian J. Genet. Plant Breed. 1979,39: 359～365.
30 Mundt, C.C., Brophy, L. S. Influence of number of host genotype units on the effectiveness of host mixtures for disease control: a modeling approach. Phytopath. 1988, 75:1141～1145.
31 Browning, J.A., Simmons, M. D., Frey, K.J. Regional deployment for conservation of oat crown rust resistance genes. In: Disease Consequences of Intensive and Extensive Culture of Field rops.Browning, J. A. (ed.). Iowa Agriculture and Home Economics Special Report 64. Iowa State University Press, Ames, Iowa, 1969, 49～56.
32 吕亮，陈其志，陈茂华等．不同水稻品种间栽控制稻瘟病的田间试验．华中农业大学学报2002，21(3)：228～230．
33 Frey, K. J., Browning, J. A. Simons M D. Management systems for host genes to control disease loss. Ann. N. Y. Acad. Sci. 1977, 255～287.
34 Wolfe, M. S. Barley diseases: maintaining the value of our varieties. In: Barley Genetics Ⅵ, vol. 2. Munck, L. (ed.). Munksgaard International Publishers, Copenhagen, Denmark, 1992, 1055～1067.
35 毛建辉，何明，何忠全．抗感品种混植对水稻主要病害的效应．植物病理学报，1991，21(2)：155～160．
36 Flor, H. Current status of the gene-for-gene concept. Annu Rev Phytopathol, 1997, 9: 275～296.
37 张冬生．《植物体细胞遗传学》，1987，复旦大学出版社．
38 刘植义，刘彭昌，周希澄等，《遗传学》，1982，人民教育出版社．
39 龙跃生，陈良碧，夏快飞等．分子标记技术及其在分离和克隆水稻基因中的应用．生物学杂志，2001，18(2)：29～32．
40 李柱刚，崔崇士，马荣才等．遗传标记在植物上的发展与应用．东北农业大学学报，2001，33(4)：313～319．
41 王和勇，陈敏，廖志化等．RFLP、RAPD、AFLP分子标记及其在植物生物技术中的应用，生物学杂志，1998，15(4)：24～26．
42 Bststein, D., White, R.L., Skolnick, M., et al. Construction of a genetic map in man use restiction fragment polymorphisms. Genet, 1980, (32): 314～3311.
43 孙雁，王云月，何月秋等．水稻抗病基因同源序列多态性与品种鉴定．种子，2001，(2)：1～2．
44 Wang, G.L., Mackill, D.J., Bonman, J.M., et al.. RFLP mapping of genes conferring complete and partial resistance to blast in a durably resistant rice cultivar. Genetics, 1994, 136: 1421～1434.
45 章琦，赵炳宇，赵开军等．普通野生稻的抗水稻白叶枯病(Xanthomonas oryzae pv.oryzae)新基因Xa-23的鉴定和分子标记．作物学报，2000，26(5)：536～542．
46 吴建利，庄杰云，柴荣耀等．水稻抗穗瘟基因的分子定位．植物病理学报，2000，30(2)：111～115．
47 聂练兵．转基因作物及其检测技术与安全性评价问题．种子科技．2001，(3)：152～154．
48 李常保．RAPD标记与作物改良．生物技术通报．1998，6：20～29．
49 赵建伟，孟金陵．植物抗病基因的克隆及其在作物遗传改良中的意义．高技术通讯，1998，58～62．
50 Wiliams, J.C.K., Kubelik, A.R.J., Livak, K,, et al.. DNA polymorphism amplified by arbitrary primes are useful as genetic markers. Nucleic Acide Reseach, 1990, 18(22):6531～6535.
51 Leister, D., Ballvora, A., Salamini, F., et al. Nature Genetics, 1996, 14: 421～429.
52 Kanazin, V., Marek, L. F., Shoemaker, R. C. Resistance gene analogs are conserved and clustered in soyabean. Proc Natl. Acad. Sci. USA, 1996, 93: 11746～11750.
53 Yu, Y. G., Buss, G. L., Maroof, M. A. S. Isolation of a super-family of candidate disease resistance genes in soyabean based on a conserved nucleotide-binding site. Proc Natl. Acad. Sci. USA, 1996, 93: 11751～11756.
54 Brune,I D., Froger, N., Pelletier, G. Development of amplified consensus genetic marker (ACGM) in Brassica napus from Arabidopsis thaliana sequences of known biological function, Genome. 1999, (42): 387-402.
55 Yu. Z. H., D. J. Mackill, J. M. Bonman, et al. Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet. 1991, 81(4): 471～476.
56 Yu, Z.H., Mackill, D.J., Bonman, J.M., et al. Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.). Theor. Appl. Genet. 1996, 93, 859～863.
57 Li-Huang Zhu, Ying Chen, Yun-Bin Xu, et al. Construction of a molecular map of rice and gene mapping using a double haploid population of a cross between Indica and Japonica varieties, Rice Genetics Newsletter, 1999, (10) 132～134.
58 Wang D.L., J. Zhu, Z.K. Li, et al.. Mapping QTLs with epistatic effects and QTLx environment interactions by mixed linear model approaches. Theor. Appl. Genet. 1999, 99:1256～1264.
59 Inukai, T., R. J. Nelson, R. S. Zeigler, et al. Allelism of blast resistance genes in near-isogenic lines of rice. Phytopathology, 1994, 84: 1278～1283.
60 T. Inukai, D. J. Mackill, P. C. Ronald, et al. Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar. Theor Appl Genet, 1999, 98: 1046～1053.
61 Wang, Z. X., Yano, M., Yamanouchi, et al. The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine- rich repeat class of plant disease esistance genes. Plant. 1999, 19, 55～64.
62 Mindrinos, M., Katagiri, F., Yu, G. L., et al. The Arabidopsis thaliana disease resistance gene RPS2 encodes a pro-tein containing a nucleotide- binding site and leucine- rich repeat. Cell, 1994, 78: 1089-1099.
63 Grant, M. R., Godiard, L., Straube, E., et al. Structure of the Arabidopsis RPM1 gene enable dual specificity di-sease resistance. Science, 1995, 269: 843～846.
64 Jones, D. A., Thomas, C. M.,Hammond- kosack, K. E., et al. Isolation of the tomato Cf- 9gene for resistance to Cladosporium fulvum by transposon tagging. Science, 1994, 266: 789～793.
65 Thomas, C. M., Jonesd, A., Parniske, M., et al. Characterization of the tomato Cf- 4gene for resistance to Cladosporium fulvum identifies sequences thatdetermine recognitional specificity in Cf- 4 and Cf- 9. Plant Cell, 1997, 9: 2209～2224.
66 Whitham, S., Dinesh- kumar, S.P., Choid, et al. The productof the tobacco mosaic virus resistance gene N: simi-larity to toll and the interieukin-1 receptor. Cell, 1994, 78:1101～1115.
67 Lawrence, G. J., Finnegen, E. J., Ayliffe, M. A., et al. The L6gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N. Plant Cell, 1995, 7:1195～1206.
68 Song, W. Y., Wang, G. L., Chen, L. L., et al. A receptor kinase- like protein encoded by the rice disease resistance gene, Xa2 1. Science, 1995, 270: 1804～1806.
69 Johal, G. S., Briggs, S. P.. Reductase activity encoded by the H m1 disease resistance gene in maize. Science, 1992,258: 985～987.
70 Cai, D., Kleine, M., Kifle, S., et al. Positional cloning of a gene for nematode resistance in sugar beet. Science, 1997, 275: 832～834.
71 李子银，陈受宜．水稻抗病基因同源序列的克隆、定位及表达．科学通报，1999，44(7)：727～733．
72 郑先武，翟文学，李晓兵等．水稻NBS-LRR类R基因同源序列．中国科学，2001，31(1)：43～51．
73 秦跟基，李万隆，陈佩度．植物抗病基因结构特征及其类似序列的研究进展．南京农业大学学报，1999，22(3)：102～107．
74 周玮斌，史晰，詹树萱等．普通野生稻中NBS同源序列的克隆和分析．复旦学报，2001，40
(5)：516～520．
75 易永图．植物抗病基因同源序列及其在抗病基因克隆与定位中的应用．生物技术通报，2002，(2)：16～20．
76 张增艳，孔凡晶，辛志勇．植物抗病基因克隆进展．生物技术通报．2000(2)：8～11
77 周益军．稻瘟病菌遗传多样性和致病性诱变研究．博士论文．2000
78 Pan, Q., wang, L., Tanisaka, T., and Ikehashi. Allelism of rice blast resistance genes in two Chinese rice cultivars, and identification of two new resistance genes. Plant Pathology, 1998, 47, 165～170.
79 McCouch, S. R., Nelson, R. J., Tohem, et al. Mapping of blast resistance genes in rice. In: Rice Blast Disease. CAB INTERNAL, Wallingford, 1994, pp. 167～182.
80 刘二明．水稻品种与稻瘟病菌的遗传多样性及稻瘟病持续控制研究．博士论文，1999．
81 张峰，宋文芹，李凌等．利用AFLP-银染法筛选与抗甘蓝黑腐病性状连锁的分子标记．南开大学学报，1999，32(3)：177～181．
82 刘二明，朱有勇，刘新民等．丘陵区水稻品种多样性混合间栽控制稻瘟病研究．作物研究，2002，16(1)：7～101．
83 王石平，刘克德，王江等．用同源序列的染色体定位寻找水稻抗病基因DNA片段．植物学报，1998，40(1)：42～50．
84 凌忠专，T．Mew，王久林等 中国水稻近等基因系的育成及其稻瘟病菌生理小种鉴别能力．中国农业科学，2000，33(4)：1～8．
85 Callard, D., Axelos, M., Mazzolini, L.. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. Plant Physiol. 1996, 112: 705～715..
86 Li, .F, E.S. Barnathan, K. Kariko. Rapid method for screening and cloning cDNA generated in differential display: amplification of Northern blot for affinity capturing of cDNAs. Nucleic Acid Research,1994, 22:1764～1765.
87 孔维文．水稻受白叶枯病菌侵染诱导的抗病相关基因的克隆与鉴定．博士论文，2001
88 Mago, R., Nair, S. and Mohan,M. Resistance gene analogues from rice: cloning, sequencing and mapping. Theor. Appl. Genet. 1999,99: 50～57.