摘要
水稻是重要的粮食作物。稻瘟病是全世界水稻主栽区发生最严重的病害之一，平均每年因稻瘟病损失的产量可供6千万人食用。防治稻瘟病最经济、安全的方法是培育含稻瘟病持久抗性基因的新品种。目前对稻瘟病抗性基因的克隆主要是通过构建双亲本遗传群体，运用图位克隆的方法实现。本研究首次应用805,158个SNP标记，对517份中国水稻地方品种资源的稻瘟病抗性进行全基因组关联分析。主要结果如下：
1.运用混合线性模型，在P<10-7水平条件下，全基因组关联分析（Genome-Wide AssociatedStudy,GWAS）共检测到126个关联位点。在籼稻群体内共检测到51个与稻瘟病抗性相关联的SNP位点；在粳稻群体内共检测到5个与稻瘟病抗性相关联的SNP位点；在总样本中共检测到72个与稻瘟病抗性相关联的SNP位点。
2.在所检测到的SNP位点中，有18个已报道位点，其中包含7个已克隆基因（Pia、Pik、Pi-1、Pik-h/Pi-54、Pik-m、Pik-p、Pi5/Pi3/Pi-i）和8个QTL位点（Pif、Pig、PiGD3、Pik-s、Pik-g(t)、Pitq5、Pitg6、Pi6）。除已知位点外，还发现18个新的SNP位点，共涉及25个尚未报道的R基因；籼稻群体中检测到17个，粳稻群体中仅检测到1个，总样本自然群体中检测到11个。其中有4个候选R基因同时在籼稻群体和总样本群体中均被检测到，1个在粳稻群体和总样本群体中被检测到。
3.利用基因功能注释（gene ontology，GO）对其它候选基因进行功能分类，划分为53个功能亚范畴。在蛋白质类型范畴中，核苷酸结合（nucleic acid binding）和水解酶（hydrolase）所占比例最大；在生物途径范畴中，代谢途径（metabolic process）所占比例最大；在代谢途径范畴中，DNA复制（DNA replication）所占比例最大；在分子功能范畴中，催化过程（catalytic activity）和连接（binding）所占比例最大。
4.本研究共选取4个显著关联位点：Chr11_6526998、Chr12_14696604、Chr12_13690289和Chr12_13032951。运用实时定量PCR（Real-time quantitative PCR，RT-PCR）技术及基因表达谱信息，对候选基因进行检测和验证。结果显示：（1）Chr11_6526998位于稻瘟病抗性基因Pia的组成基因Os11g0225100的编码区；（2）Chr12_14696604位于PiGD3（QTL）附近，研究发现基因Os12g0438300（含有NB-ARC和LRR结构域）很可能是PiGD3的最优候选基因；（3）对于Chr12_13690289关联位点，基因Os12g0424700和基因Os12g0427000可能相互协作，共同完成稻瘟病抗性调控；（4）对于Chr12_13032951关联位点，基因Os12g0416300、Os12g0417100和Os12g0417600被视为最优候选基因。
5.候选基因单倍型分析表明大部分SNPs变异均属于非同义突变。
6.本研究证实GWAS能够作为一种高效率挖掘抗性候选基因的方法。
Rice is a vital and stable grain food, but rice blast disease is one of the most serious and recurrentproblems in rice-growing regions. Every year the rice capable of feeding60million people is destroyedby rice blast disease. Hence, cultivating rice varieties with highly efficient, durable resistance to blast isstill the most economically feasible and environmentally sound management approach in mostblast-prone rice ecosystems. Most previous studies cloned resistance genes were based on linkagemapping using bi-parental populations. Here, we extensively examined blast resistance in agenome-wide association study (GWAS) based on genotyping805,158SNPs variants across517diverse rice accessions. The main results were as follows:
1. GWAS identified126associated loci at P<10-7in the compressed MLM. A total of51associatedSNPs were identified in indica panel. Only five associated loci were detected in japonica panel. AGWAS in the full panel was higher power with a total of72SNPs.
2. Searching the flanking regions of the associated SNPs, we revealed18regions co-localized, withseven known cloned genes (Pia, Pik, Pi-1, Pik-h/Pi-54, Pik-m, Pik-p, Pi5/Pi3/Pi-i), and eight QTLs (Pif,Pig, PiGD3, Pik-s, Pik-g(t), Pitq5, Pitg6, Pi6). In addition to the loci associated with the known, wealso found another18new loci referring to25previously unreported candidate genes, which describedas disease resistance protein and showed potential function for rice blast as R genes, separately17inindica panel, one in japonica panel and11in the full panel. Of25candidate genes，four simultaneouslyfound in indica and the full panel, and one simultaneously in japonica and the full panel.
3. According to gene ontology (GO) analysis, the others encoding proteins involved53classifications.In the protein class, nucleic acid binding and hybrolase were the main. In the metabolic pathways, DNAreplication was major. In the biological process, metabolic process was the most. In the molecularfunction, catalytic activity and binding were the largest proportion.
4. We chose four associated SNPs to identify the candidate genes, referring to Chr11_6526998,Chr12_14696604, Chr12_13690289and Chr12_13032951. Through RT-PCR and expression profilesfrom microarray data, we observed:(1) Chr11_6526998was within the coding domain of the geneOs11g0225100, one of the rice Pia-blast resistance gene.(2) A signal, Chr12_14696604, was detectedaround the QTL PiGD3. And, our study identified the gene Os12g0438300, a disease resistance proteincontaining NB-ARC and leucine-rich repeat domain, as the main candidate gene of PiGD3.(3) Forassociated SNP, Chr12_13690289, the newly discovered R genes, Os12g0424700and Os12g0427000were presumed to be required for the full function of this locus.(4) In addition, for Chr12_13032951,the region without R genes, Os12g0416300, Os12g0417100, and Os12g0417600were considered themost likely candidate genes for this region.
5. Haplotype analysis around the candidate genes reflected that most of the SNPs were non-synonymousmutation.
6. Our results further confirmed that GWAS is a powerful complementary approach for dissecting thequantitative disease resistant genes to traditional QTL mapping.
引文
1.邓其明,周鹏,林琳,等.水稻稻瘟病抗性基因研究进展及其在育种上的应用[J].安徽农业科学,2009,37(4):1489-1492
2.鄂志国,王磊.水稻抗病性基因的克隆和功能研究进展[J].遗传,2009,31(10):999-1005
3.高清松,杨泽峰,徐辰武.水稻基因组进化的研究进展[J].扬州大学学报:农业与生命科学版,2009,30(2):34-44
4.李林.结合连锁与关联分析剖析高油玉米遗传机理[博士学位论文].北京:中国农业大学,2010
5.刘春明,于占洋,朱祯,等.豌豆外源凝集素基因的克隆及序列分析[J].遗传学报,1995,22(4):302-306
6.刘仁虎,孟金陵. MapDraw在Excel中绘制遗传连锁图的宏[J].遗传,2003,25(3):317-321
7.陆凡,范永坚.江苏省稻瘟病菌有性态的研究[J].菌物系统,2001,20(1):122-128
8.清泽茂久,凌忠专.用日本菌系鉴定中国鉴别品种的稻瘟病抗性基因[J].中国科学: B辑,1984,1:44-52
9.全国稻瘟病生理小种联合试验组.我国稻瘟病菌生理小种研究[J].植物病理学报,1980,10(2):71-81
10.山崎义人,高坂淖尔编著.凌忠专,孙昌其译.林世成校.稻瘟病与抗病育种[M].北京:农业出版社,1990:353
11.沈锦骅,凌忠专,倪丕冲,等.中日两套鉴别品种的鉴别力研究[J].作物学报,1986,12(3):163-170
12.沈瑛,金敏忠.我国稻瘟病菌有性态的研究[J].中国农业科学,1994,27(1):25-29
13.王荣焕,王天宇,黎裕.关联分析在作物种质资源分子评价中的应用[J].植物遗传资源学报,2007,8(3):366-372
14.杨红,储昭晖,傅晶,等.抗稻瘟病主效QTL rbr2是Pib的等位基因[J].分子植物育种,2008,6(2):213-219
15.杨勤忠,林菲,冯淑杰,等.水稻稻瘟病抗性基因的分子定位及克隆研究进展[J].中国农业科学,2009,42(5):1601-1615
16. Agrama H A, Yan W G, Jia M, et al. Genetic structure associated with diversity and geographicdistribution in the USDA rice world collection[J]. Engineering,2010,2(4).
17. Aranzana M J, Kim S, Zhao K, et al. Genome-wide association mapping in Arabidopsis identifiespreviously known flowering time and pathogen resistance genes[J]. PLoS genetics,2005,1(5): e60
18. Ashikawa I, Hayashi N, Yamane H, et al. Two adjacent nucleotide-binding site–leucine-rich repeatclass genes are required to confer Pikm-specific rice blast resistance[J]. Genetics,2008,180(4):2267-2276
19. Atkins J G, Robert A L, Adair C R, et al. An international set of rice varieties for differentiatingraces of Piricularia oryzae[J]. Phytopathology,1967,57(3):297-301
20. Atwell S, Huang Y S, Vilhjálmsson B J, et al. Genome-wide association study of107phenotypes inArabidopsis thaliana inbred lines[J]. Nature,2010,465(7298):627-631
21. Austin M J, Muskett P, Kahn K, et al. Regulatory role of SGT1in early R gene-mediated plantdefenses[J]. Science,2002,295(5562):2077-2080
22. Bai J, Pennill L A, Ning J, et al. Diversity in nucleotide binding site–leucine-rich repeat genes incereals[J]. Genome research,2002,12(12):1871-1884
23. Barrett J C, Fry B, Maller J, et al. Haploview: analysis and visualization of LD and haplotypemaps[J]. Bioinformatics,2005,21(2):263-265
24. Belkhadir Y, Subramaniam R, Dangl J L. Plant disease resistance protein signaling: NBS–LRRproteins and their partners[J]. Current opinion in plant biology,2004,7(4):391-399
25. Bergelson J, Roux F. Towards identifying genes underlying ecologically relevant traits inArabidopsis thaliana[J]. Nature Reviews Genetics,2010,11(12):867-879
26. Bernardo R. Molecular markers and selection for complex traits in plants: learning from the last20years[J]. Crop Science,2008,48(5):1649-1664
27. Blanc G, Hokamp K, Wolfe K H. A recent polyploidy superimposed on older large-scaleduplications in the Arabidopsis genome[J]. Genome research,2003,13(2):137-144
28. B hnert H U, Fudal I, Dioh W, et al. A putative polyketide synthase/peptide synthetase fromMagnaporthe grisea signals pathogen attack to resistant rice[J]. The Plant Cell Online,2004,16(9):2499-2513
29. Bourett T M, Howard R J. Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea[J]. Protoplasma,1992,168(1-2):20-26
30. Bryan G T, Wu K S, Farrall L, et al. A single amino acid difference distinguishes resistant andsusceptible alleles of the rice blast resistance gene Pi-ta[J]. The Plant Cell Online,2000,12(11):2033-2045
31. Buckler E S, Holland J B, Bradbury P J, et al. The genetic architecture of maize flowering time[J].Science,2009,325(5941):714-718
32. Büschges R, Hollricher K, Panstruga R, et al. The Barley Mlo Gene: A Novel Control Element ofPlant Pathogen Resistance[J]. Cell,1997,88(5):695-705
33. Century K S, Holub E B, Staskawicz B J. NDR1, a locus of Arabidopsis thaliana that is requiredfor disease resistance to both a bacterial and a fungal pathogen[J]. Proceedings of the NationalAcademy of Sciences,1995,92(14):6597-6601
34. Chauhan R, Farman M, Zhang H B, et al. Genetic and physical mapping of a rice blast resistancelocus, Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39of Magnaporthe grisea[J].Molecular Genetics and Genomics,2002,267(5):603-612
35. Chen H L, Chen B T, Zhang D P, et al. Pathotypes of Pyricularia grisea in rice fields of central andsouthern China[J]. Plant Disease,2001,85(8):843-850
36. Chen X, Shang J, Chen D, et al. A β-lectin receptor kinase gene conferring rice blast resistance[J].The Plant Journal,2006,46(5):794-804
37. Cho R J, Mindrinos M, Richards D R, et al. Genome-wide mapping with biallelic markers inArabidopsis thaliana[J]. Nature genetics,1999,23(2):203-207
38. Chun S J, Lee Y H. Genetic analysis of a mutation on appressorium formation in Magnaporthegrisea[J]. FEMS microbiology letters,1999,173(1):133-137
39. Davis E E, Zhang Q, Liu Q, et al. TTC21B contributes both causal and modifying alleles across theciliopathy spectrum[J]. Nature genetics,2011,43(3):189-196
40. Dean R A, Talbot N J, Ebbole D J, et al. The genome sequence of the rice blast fungusMagnaporthe grisea[J]. Nature,2005,434(7036):980-986
41. Deng Y, Zhu X, Shen Y, et al. Genetic characterization and fine mapping of the blast resistancelocus Pigm (t) tightly linked to Pi2and Pi9in a broad-spectrum resistant Chinese variety[J].Theoretical and applied genetics,2006,113(4):705-713
42. Dufresne M, Osbourn A E. Definition of tissue-specific and general requirements for plantinfection in a phytopathogenic fungus[J]. Molecular plant-microbe interactions,2001,14(3):300-307
43. Duijvesteijn N, Knol E F, Merks J W M, et al. A genome-wide association study on androstenonelevels in pigs reveals a cluster of candidate genes on chromosome6[J]. BMC genetics,2010,11(1):42
44. Ebana K, Kojima Y, Fukuoka S, et al. Development of mini core collection of Japanese ricelandrace[J]. Breeding Science,2008,58(3):281-291
45. Ebanat K, Yonemaru J, Fukuoka S, et al. Genetic structure revealed by a whole-genomesingle-nucleotide polymorphism survey of diverse accessions of cultivated Asian rice (Oryzasaliva L.)[J]. Breeding science,2010,60(4)
46. Edwards A O, Ritter R, Abel K J, et al. Complement factor H polymorphism and age-relatedmacular degeneration[J]. Science,2005,308(5720):421-424
47. Emmett R W, Parbery D G. Appressoria[J]. Annual Review of Phytopathology,1975,13(1):147-165
48. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the softwareSTRUCTURE: a simulation study[J]. Molecular ecology,2005,14(8):2611-2620
49. Falush D, Stephens M, Pritchard J K. Inference of population structure using multilocus genotypedata: linked loci and correlated allele frequencies[J]. Genetics,2003,164(4):1567-1587
50. Fearnhead P, Donnelly P. Estimating recombination rates from population genetic data[J]. Genetics,2001,159(3):1299-1318
51. Felsenstein J. PHYLIP: Phylogeny Inference Package (Version3.2)[J]. Cladistics,1989,5:164-166
52. Fernie A R, Schauer N. Metabolomics-assisted breeding: a viable option for crop improvement[J].Trends in Genetics,2009,25(1):39-48
53. Feuk L, Carson A R, Scherer S W. Structural variation in the human genome[J]. Nature ReviewsGenetics,2006,7(2):85-97
54. Flint-Garcia S A, Thornsberry J M, IV B. Structure of Linkage Disequilibrium in Plants[J]. AnnualReview of Plant Biology,2003,54(1):357-374
55. Flor H H. Current status of the gene-for-gene concept[J]. Annual review of phytopathology,1971,9(1):275-296
56. Freedman M L, Reich D, Penney K L, et al. Assessing the impact of population stratification ongenetic association studies[J]. Nature genetics,2004,36(4):388-393
57. Freeman J L, Perry G H, Feuk L, et al. Copy number variation: new insights in genome diversity[J].Genome research,2006,16(8):949-961
58. Fukuoka S, Okuno K. QTL analysis and mapping of pi21, a recessive gene for field resistance torice blast in Japanese upland rice[J]. Theoretical and Applied Genetics,2001,103(2-3):185-190
59. Fukuoka S, Saka N, Koga H, et al. Loss of function of a proline-containing protein confers durabledisease resistance in rice[J]. Science,2009,325(5943):998-1001
60. Gaut B S, Long A D. The lowdown on linkage disequilibrium[J]. The Plant Cell Online,2003,15(7):1502-1506
61. Gilmour J. Octal notation for designating physiologic races of plant pathogens[J]. Nature,1973,620
62. Goff S A, Ricke D, Lan T H, et al. A draft sequence of the rice genome (Oryza sativa L. ssp.japonica)[J]. Science,2002,296(5565):92-100
63. Goidts V, Cooper D N, Armengol L, et al. Complex patterns of copy number variation at sites ofsegmental duplications: an important category of structural variation in the human genome[J].Human genetics,2006,120(2):270-284
64. Gu X, Feng C, Ma L, et al. Genome-wide association study of body weight in chicken F2resourcepopulation[J]. PLoS One,2011,6(7): e21872
65. Hamer J E, Howard R J, Chumley F G, et al. A mechanism for surface attachment in spores of aplant pathogenic fungus[J]. Science,1988,239(4837):288-290
66. Hardy O J, Vekemans X. SPAGeDi: a versatile computer program to analyse spatial geneticstructure at the individual or population levels[J]. Molecular ecology notes,2002,2(4):618-620
67. Hayashi K, Yoshida H. Refunctionalization of the ancient rice blast disease resistance gene Pit bythe recruitment of a retrotransposon as a promoter[J]. The Plant Journal,2009,57(3):413-425
68. Hayes B J, Bowman P J, Chamberlain A J, et al. Invited review: Genomic selection in dairy cattle:Progress and challenges[J]. Journal of dairy science,2009,92(2):433-443
69. Heath M C, Valent B, Howard R J, et al. Correlations between cytologically detected plant-fungalinteractions and pathogenicity of Magnaporthe grisea toward weeping lovegrass[J].Phytopathology,1990b,80(12):1382-1386
70. Heath M C, Valent B, Howard R J, et al. Interactions of two strains of Magnaporthe grisea withrice, goosegrass, and weeping lovegrass[J]. Canadian Journal of Botany,1990a,68(8):1627-1637
71. Helgadottir A, Thorleifsson G, Manolescu A, et al. A common variant on chromosome9p21affects the risk of myocardial infarction[J]. Science,2007,316(5830):1491-1493
72. Helgason A, Yngvadóttir B, Hrafnkelsson B, et al. An Icelandic example of the impact ofpopulation structure on association studies[J]. Nature genetics,2005,37(1):90-95
73. Herbert A, Gerry N P, McQueen M B, et al. A common genetic variant is associated with adult andchildhood obesity[J]. Science,2006,312(5771):279-283
74. Hinds D A, Stokowski R P, Patil N, et al. Matching strategies for genetic association studies instructured populations[J]. The American Journal of Human Genetics,2004,74(2):317-325
75. Hirschhorn J N, Daly M J. Genome-wide association studies for common diseases and complextraits[J]. Nature Reviews Genetics,2005,6(2):95-108
76. Hittalmani S, Parco A, Mew T V, et al. Fine mapping and DNA marker-assisted pyramiding of thethree major genes for blast resistance in rice[J]. Theoretical and Applied Genetics,2000,100(7):1121-1128
77. Howard R J, Ferrari M A, Roach D H, et al. Penetration of hard substrates by a fungus employingenormous turgor pressures[J]. Proceedings of the National Academy of Sciences,1991,88(24):11281-11284
78. Howard R J, Ferrari M A. Role of melanin in appressorium function[J]. Experimental Mycology,1989,13(4):403-418
79. Howard R J, Valent B. Breaking and entering: host penetration by the fungal rice blast pathogenMagnaporthe grisea[J]. Annual Reviews in Microbiology,1996,50(1):491-512
80. Huang X, Wei X, Sang T, et al. Genome-wide association studies of14agronomic traits in ricelandraces[J]. Nature genetics,2010,42(11):961-967.
81. Huang X, Zhao Y, Wei X, et al. Genome-wide association study of flowering time and grain yieldtraits in a worldwide collection of rice germplasm[J]. Nature genetics,2012,44(1):32-39.
82. Hulbert S H, Webb C A, Smith S M, et al. Resistance gene complexes: evolution and utilization[J].Annual review of phytopathology,2001,39(1):285-312
83. Hunter D J. Gene-environment interactions in human diseases[J]. Nature Reviews Genetics,2005,6(4):287-298.
84. Ikram M A, Seshadri S, Bis J C, et al. Genomewide association studies of stroke[J]. New EnglandJournal of Medicine,2009,360(17):1718-1728
85. Imbe T, Matsumoto S. Inheritance of resistance of rice varieties to the blast fungus strains virulentto the variety[J]. Japanese Journal of Breeding,1985,35
86. Imbe T, Oba S, Yanoria M J T, et al. A new gene for blast resistance in rice cultivar, IR24[J]. RiceGenet. Newsl,1997,14:60-62
87. Inoue I, Namiki F, Tsuge T. Plant colonization by the vascular wilt fungus Fusarium oxysporumrequires FOW1, a gene encoding a mitochondrial protein[J]. The Plant Cell Online,2002,14(8):1869-1883
88. International HapMap Consortium. A haplotype map of the human genome[J]. Nature,2005,437(7063):1299-1320.
89. Izawall T, Iwasakizl M. Identifcation of a RFLP Marker Tightly Linked to the Panicle BlastResistance Gene, Pb1, in Rice[J]. Breeding science,2000,50:183-188
90. Jakobsson M, Rosenberg N A. CLUMPP: a cluster matching and permutation program for dealingwith label switching and multimodality in analysis of population structure[J]. Bioinformatics,2007,23(14):1801-1806
91. Jia G, Huang X, Zhi H, et al. A haplotype map of genomic variations and genome-wide associationstudies of agronomic traits in foxtail millet (Setaria italica)[J]. Nature genetics,2013,45(8):957-961
92. Jia Y, McAdams S A, Bryan G T, et al. Direct interaction of resistance gene and avirulence geneproducts confers rice blast resistance[J]. The EMBO Journal,2000,19(15):4004-4014
93. Johal G S, Briggs S P. Reductase activity encoded by the HM1disease resistance gene in maize[J].Science,1992,258(5084):985-987
94. Jonsson J J, Weissman S M. From mutation mapping to phenotype cloning[J]. Proceedings of theNational Academy of Sciences of the United States of America,1995,92(1):83
95. Juran B D, Lazaridis K N. Genomics in the post-GWAS era[C]. Seminars in liver disease. NIHPublic Access,2011,31(2):215.
96. Kang H M, Zaitlen N A, Wade C M, et al. Efficient control of population structure in modelorganism association mapping[J]. Genetics,2008,178(3):1709-1723
97. Kato M, Nakamura Y, Tsunoda T. An algorithm for inferring complex haplotypes in a region ofcopy-number variation[J]. The American Journal of Human Genetics,2008,83(2):157-169
98. Ke X, Hunt S, Tapper W, et al. The impact of SNP density on fine-scale patterns of linkagedisequilibrium[J]. Human Molecular Genetics,2004,13(6):577-588
99. Keen N T. Gene-for-gene complementarity in plant-pathogen interactions[J]. Annual review ofgenetics,1990,24(1):447-463
100. Kettunen J, Tukiainen T, Sarin A P, et al. Genome-wide association study identifies multiple lociinfluencing human serum metabolite levels[J]. Nature genetics,2012,44(3):269-276
101. Kump K L, Bradbury P J, Wisser R J, et al. Genome-wide association study of quantitativeresistance to southern leaf blight in the maize nested association mapping population[J]. Naturegenetics,2011,43(2):163-168
102. Lee S K, Song M Y, Seo Y S, et al. Rice Pi5-mediated resistance to Magnaporthe oryzae requiresthe presence of two coiled-coil–nucleotide-binding–leucine-rich repeat genes[J]. Genetics,2009,181(4):1627-1638
103. Li H, Peng Z, Yang X, et al. Genome-wide association study dissects the genetic architecture of oilbiosynthesis in maize kernels[J]. Nature genetics,2013,45(1):43-50
104. Lin F, Chen S, Que Z, et al. The blast resistance gene Pi37encodes a nucleotide bindingsite–leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome1[J]. Genetics,2007,177(3):1871-1880
105. Lincoln S E, Daly M J, Lander E S. Mapping genes controlling quantitative traits usingMAPMAKER/QTL version1.1: a tutorial and reference manual[J]. Whitehead Institute forBiometrical Research, Cambridge, Mass,1993,6-47.
106. Lindblad-Toh K, Winchester E, Daly M J, et al. Large-scale discovery and genotyping ofsingle-nucleotide polymorphisms in the mouse[J]. Nature genetics,2000,24(4):381-386
107. Ling Z Z, Mew T V, Wang J L, et al. Development of near-isogenic lines as internationaldifferentials of the blast pathogen[J]. International Rice Research Notes,1995,20
108. Ling Z Z, Mew T, Wang J L, et al. Development of Chinese near isogenic lines of rice and theirdifferentiating ability of pathogenic races of blast fungus[J]. Chin Agric Sci,2001,1:50-56
109. Liu B, Zhang S, Zhu X, et al. Candidate defense genes as predictors of quantitative blast resistancein rice[J]. Molecular plant-microbe interactions,2004,17(10):1146-1152
110. Liu G, Lu G, Zeng L, et al. Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), arephysically linked on rice chromosome6[J]. Molecular Genetics and Genomics,2002,267(4):472-480
111. Liu J, Liu X, Dai L, et al. Recent progress in elucidating the structure, function and evolution ofdisease resistance genes in plants[J]. Journal of Genetics and Genomics,2007,34(9):765-776
112. Liu K J, Muse S V. Powermarker: Integrated analysis environment for genetic maker date[J].Bioinformatics,2005,21:2128-2129
113. Liu X, Lin F, Wang L, et al. The in silico map-based cloning of Pi36, a ricecoiled-coil–nucleotide-binding site–leucine-rich repeat gene that confers race-specific resistance tothe blast fungus[J]. Genetics,2007,176(4):2541-2549
114. Loiselle B A, Sork V L, Nason J, et al. Spatial genetic structure of a tropical understory shrub,Psychotria officinalis (Rubiaceae)[J]. American Journal of Botany,1995:1420-1425.
115. Lu X, Wang L, Chen S, et al. Genome-wide association study in Han Chinese identifies four newsusceptibility loci for coronary artery disease[J]. Nature genetics,2012,44(8):890-894
116. Mackill D J, Bonman J M. Inheritance of blast resistance in near-isogenic lines of rice[J].Phytopathology,1992,82(7):746-749
117. Mai M D, Sahana G, Christiansen F B, et al. A genome-wide association study for milk productiontraits in Danish Jersey cattle using a50K single nucleotide polymorphism chip[J]. Journal ofanimal science,2010,88(11):3522-3528
118. Manicacci D, Camus-Kulandaivelu L, Fourmann M, et al. Epistatic interactions between Opaque2transcriptional activator and its target gene CyPPDK1control kernel trait variation in maize[J].Plant physiology,2009,150(1):506-520
119. Marchini J, Cardon L R, Phillips M S, et al. The effects of human population structure on largegenetic association studies[J]. Nature genetics,2004,36(5):512-517
120. Martin G B, Bogdanove A J, Sessa G. Understanding the functions of plant disease resistanceproteins[J]. Annual review of plant biology,2003,54(1):23-61
121. Martin G B, Brommonschenkel S H, Chunwongse J, et al. Map-based cloning of a protein kinasegene conferring disease resistance in tomato[J]. Science,1993,262:1432-1432
122. Mather K A, Caicedo A L, Polato N R, et al. The extent of linkage disequilibrium in rice (Oryzasativa L.)[J]. Genetics,2007,177(4):2223-2232
123. Mauricio R, Stahl E A, Korves T, et al. Natural selection for polymorphism in the diseaseresistance gene Rps2of Arabidopsis thaliana[J]. Genetics,2003,163(2):735-746
124. McCouch S R, Zhao K, Wright M, et al. Development of genome-wide SNP assays for rice [J].Breeding Science，2010,60(5):524-535
125. McCullagh P. Generalized linear models[J]. European Journal of Operational Research,1984,16(3):285-292
126. McHale L, Tan X, Koehl P, et al. Plant NBS-LRR proteins: adaptable guards[J]. Genome Biol,2006,7(4):212
127. McNally K L, Childs K L, Bohnert R, et al. Genomewide SNP variation reveals relationshipsamong landraces and modern varieties of rice[J]. Proceedings of the National Academy of Sciences,2009,106(30):12273-12278
128. Meyers B C, Dickerman A W, Michelmore R W, et al. Plant disease resistance genes encodemembers of an ancient and diverse protein family within the nucleotide-binding superfamily[J].The Plant Journal,1999,20(3):317-332
129. Narusaka M, Shirasu K, Noutoshi Y, et al. RRS1and RPS4provide a dual Resistance‐genesystem against fungal and bacterial pathogens[J]. The Plant Journal,2009,60(2):218-226
130. Nejentsev S, Walker N, Riches D, et al. Rare variants of IFIH1, a gene implicated in antiviralresponses, protect against type1diabetes[J]. Science,2009,324(5925):387-389
131. Okuyama Y, Kanzaki H, Abe A, et al. A multifaceted genomics approach allows the isolation of therice Pia-blast resistance gene consisting of two adjacent NBS‐LRR protein genes[J]. The PlantJournal,2011,66(3):467-479
132. Pan Q, Liu Y S, Budai-Hadrian O, et al. Comparative genetics of nucleotide binding site-leucinerich repeat resistance gene homologues in the genomes of two dicotyledons: tomato andArabidopsis[J]. Genetics,2000,155(1):309-322
133. Panaud O, Chen X, McCouch S R. Development of microsatellite markers and characterization ofsimple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)[J]. Molecular and GeneralGenetics MGG,1996,252(5):597-607
134. Parker J E, Holub E B, Frost L N, et al. Characterization of eds1, a mutation in Arabidopsissuppressing resistance to Peronospora parasitica specified by several different RPP genes[J]. ThePlant Cell Online,1996,8(11):2033-2046
135. Paterson A H, Chapman B A, Kissinger J C, et al. Many gene and domain families haveconvergent fates following independent whole-genome duplication events in Arabidopsis, Oryza,Saccharomyces and Tetraodon[J]. Trends in Genetics,2006,22(11):597-602
136. Patterson N, Price A L, Reich D. Population structure and eigenanalysis[J]. PLoS genetics,2006,2(12): e190
137. Payne G, Ahl P, Moyer M, et al. Isolation of complementary DNA clones encodingpathogenesis-related proteins P and Q, two acidic chitinases from tobacco[J]. Proceedings of theNational Academy of Sciences,1990,87(1):98-102
138. Peng Y L, Shishiyama J. Temporal sequence of cytological events in rice leaves infected withPyricularia oryzae[J]. Canadian journal of botany,1988,66(4):730-735
139. Poland J A, Bradbury P J, Buckler E S, et al. Genome-wide nested association mapping ofquantitative resistance to northern leaf blight in maize[J]. Proceedings of the National Academy ofSciences,2011,108(17):6893-6898
140. Price A L, Patterson N J, Plenge R M, et al. Principal components analysis corrects forstratification in genome-wide association studies[J]. Nature genetics,2006,38(8):904-909
141. Primmer C R, Borge T, Lindell J, et al. Single‐nucleotide polymorphism characterization inspecies with limited available sequence information: high nucleotide diversity revealed in the aviangenome[J]. Molecular Ecology,2002,11(3):603-612
142. Pritchard J K, Stephens M, Donnelly P. Inference of population structure using multilocusgenotype data[J]. Genetics,2000,155(2):945-959
143. Qu S, Liu G, Zhou B, et al. The broad-spectrum blast resistance gene Pi9encodes anucleotide-binding site–leucine-rich repeat protein and is a member of a multigene family in rice[J].Genetics,2006,172(3):1901-1914
144. Rabinowitz D, Laird N. A unified approach to adjusting association tests for population admixturewith arbitrary pedigree structure and arbitrary missing marker information[J]. Human heredity,2000,50(4):211-223
145. Rafalski A, Morgante M. Corn and humans: recombination and linkage disequilibrium in twogenomes of similar size[J]. TRENDS in Genetics,2004,20(2):103-111
146. Raychaudhuri S. Mapping rare and common causal alleles for complex human diseases[J]. Cell,2011,147(1):57-69
147. Reiner A P, Ziv E, Lind D L, et al. Population structure, admixture, and aging-related phenotypes inAfrican American adults: the Cardiovascular Health Study[J]. The American Journal of HumanGenetics,2005,76(3):463-477
148. Remington D L, Thornsberry J M, Matsuoka Y, et al. Structure of linkage disequilibrium andphenotypic associations in the maize genome[J]. Proceedings of the National Academy of Sciences,2001,98(20):11479-11484
149. Rohlf F J. NTSYS-pc: numerical taxonomy and multivariate analysis system[M]. AppliedBiostatistics,1992
150. Salvi S, Tuberosa R. To clone or not to clone plant QTLs: present and future challenges[J]. Trendsin plant science,2005,10(6):297-304
151. Sanna S, Jackson A U, Nagaraja R, et al. Common variants in the GDF5-UQCC region areassociated with variation in human height[J]. Nature genetics,2008,40(2):198-203
152. SAS Institute. SAS/STAT user's guide: version6[M]. Sas Inst,1990
153. Sasaki R. Existence of strains in rice blast fungus[J]. Japanese Journal of Plant Protection,1922,9:631-644
154. Savolainen O, Langley C H, Lazzaro B P, et al. Contrasting patterns of nucleotide polymorphism atthe alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsisthaliana[J]. Molecular Biology and Evolution,2000,17(4):645-655
155. Saxena R, Voight B F, Lyssenko V, et al. Genome-wide association analysis identifies loci for type2diabetes and triglyceride levels[J]. Science,2007,316(5829):1331-1336
156. Sesma A, Osbourn A E. The rice leaf blast pathogen undergoes developmental processes typical ofroot-infecting fungi[J]. Nature,2004,431(7008):582-586
157. Shang J, Tao Y, Chen X, et al. Identification of a new rice blast resistance gene, Pid3, bygenomewide comparison of paired nucleotide-binding site–leucine-rich repeat genes and theirpseudogene alleles between the two sequenced rice genomes[J]. Genetics,2009,182(4):1303-1311
158. Sharma T R, Madhav M S, Singh B K, et al. High-resolution mapping, cloning and molecularcharacterization of the Pi-khgene of rice, which confers resistance to Magnaporthe grisea[J].Molecular Genetics and Genomics,2005,274(6):569-578
159. Shindo C, Aranzana M J, Lister C, et al. Role of FRIGIDA and FLOWERING LOCUS C indetermining variation in flowering time of Arabidopsis[J]. Plant Physiology,2005,138(2):1163-1173
160. Shirasu K, Lahaye T, Tan M W, et al. A Novel Class of Eukaryotic Zinc-Binding Proteins IsRequired for Disease Resistance Signaling in Barley and Development in C. elegans [J]. Cell,1999,99(4):355-366
161. Shuyuan S. The Directional Selection and Stabilizing Selection from the Interaction between RiceVariety and Blast Fungus Magnaporthe grisea [J]. Acta Phytopathologica Sinica,1999,1
162. Simonich M T, Innes R W. A disease resistance gene in Arabidopsis with specificity for theavrPph3gene of Pseudomonas syringae pv. phaseolicola[J]. Molecular plant-microbe interactions:MPMI,1994,8(4):637-640
163. SPSS inc. SYSTAT ver.7, The system for statistics[J].1997
164. Stahl E A, Dwyer G, Mauricio R, et al. Dynamics of disease resistance polymorphism at the Rpm1locus of Arabidopsis[J]. Nature,1999,400(6745):667-671
165. Staskawicz B J. Genetics of plant-pathogen interactions specifying plant disease resistance[J].Plant Physiology,2001,125(1):73-76
166. Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction frompopulation genotype data[J]. The American Journal of Human Genetics,2003,73(5):1162-1169
167. Stinchcombe J R, Weinig C, Ungerer M, et al. A latitudinal cline in flowering time in Arabidopsisthaliana modulated by the flowering time gene FRIGIDA[J]. Proceedings of the National Academyof Sciences of the United States of America,2004,101(13):4712-4717
168. Syv nen A C. Accessing genetic variation: genotyping single nucleotide polymorphisms[J]. NatureReviews Genetics,2001,2(12):930-942
169. Tamura K, Peterson D, Peterson N, et al. MEGA5: molecular evolutionary genetics analysis usingmaximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecularbiology and evolution,2011,28(10):2731-2739
170. Tanaka T, Antonio B A, Kikuchi S, et al. The rice annotation project database (RAP-DB):2008update[J]. Nucleic acids research,2008,36(Supp1): D1028-D1033.
171. Thornsberry J M, Goodman M M, Doebley J, et al. Dwarf8polymorphisms associate withvariation in flowering time[J]. Nature genetics,2001,28(3):286-289
172. Tian D, Araki H, Stahl E, et al. Signature of balancing selection in Arabidopsis[J]. Proceedings ofthe National Academy of Sciences,2002,99(17):11525-11530
173. Tomlinson E, Fu L, John L, et al. Transgenic mice expressing human fibroblast growth factor-19display increased metabolic rate and decreased adiposity[J]. Endocrinology,2002,143(5):1741-1747
174. Tomlinson I P M, Carvajal-Carmona L G, Dobbins S E, et al. Multiple common susceptibilityvariants near BMP pathway loci GREM1, BMP4, and BMP2explain part of the missing heritabilityof colorectal cancer[J]. PLoS genetics,2011,7(6): e1002105
175. Valent B, Farrall L, Chumley F G. Magnaporthe grisea genes for pathogenicity and virulenceidentified through a series of backcrosses[J]. Genetics,1991,127(1):87-101
176. Valent B. Rice blast as a model system for plant pathology[J]. Phytopathology,1990,80(1):33-36
177. Van Der Biezen E A, Jones J D G. Plant disease-resistance proteins and the gene-for-geneconcept[J]. Trends in biochemical sciences,1998,23(12):454-456
178. Variar M. Pathogenic variation in Magnaporthe grisea and breeding for blast resistance in India[J].Japan International Research Center for Agricultural Sciences Working Report,2007(53):87-95
179. Wang C H, Zheng X M, Xu Q, et al. Genetic diversity and classification of Oryza sativa withemphasis on Chinese rice germplasm[J]. Heredity,2013
180. Wang G L, Mackill D J, Bonman J M, et al. RFLP mapping of genes conferring complete andpartial resistance to blast in a durably resistant rice cultivar[J]. Genetics,1994,136(4):1421-1434
181. Wang K, Diskin S J, Zhang H, et al. Integrative genomics identifies LMO1as a neuroblastomaoncogene[J]. Nature,2011,469(7329):216-220
182. Wang K, Li M, Bucan M. Pathway-based approaches for analysis of genome-wide associationstudies [J]. The American Journal of Human Genetics,2007,81(6):1278-1283
183. Wang S C, Zheng Z B, Basten C J, et al. QTL cartographer for windows version2.5[J]. Departmentof Statistics, North Carolina State University, Raleigh, North Carolina,2005
184. Wang W Y S, Barratt B J, Clayton D G, et al. Genome-wide association studies: theoretical andpractical concerns[J]. Nature Reviews Genetics,2005,6(2):109-118
185. Wang Z X, Yano M, Yamanouchi U, et al. The Pib gene for rice blast resistance belongs to thenucleotide binding and leucine‐rich repeat class of plant disease resistance genes[J]. The PlantJournal,1999,19(1):55-64
186. Wasano N, Ohgushi A, Ohba M. Mannose-specific lectin activity of parasporal proteins from alepidoptera-specific Bacillus thuringiensis strain[J]. Current microbiology,2003,46(1):0043-0046
187. Weber A, Clark R M, Vaughn L, et al. Major regulatory genes in maize contribute to standingvariation in teosinte (Zea mays ssp. parviglumis)[J]. Genetics,2007,177(4):2349-2359
188. Widawsky D A, O'Toole J C. Prioritizing the rice biotechnology research agenda for easternIndia[M]. Rockefeller Foundation,1990
189. Xiao S, Ellwood S, Calis O, et al. Broad-spectrum mildew resistance in Arabidopsis thalianamediated by RPW8[J]. Science,2001,291(5501):118-120
190. Xu J, Mo Z, Ye D, et al. Genome-wide association study in Chinese men identifies two newprostate cancer risk loci at9q31.2and19q13.4[J]. Nature genetics,2012,44(11):1231-1235
191. Yamada M, Kiyosawa S, Yamaguchi T, et al. Proposal of a new method for differentiating races ofPyricularia oryzae Cavara in Japan[J]. Annals of the Phytopathological Society of Japan,1976
192. Yan J, Shah T, Warburton M L, et al. Genetic characterization and linkage disequilibriumestimation of a global maize collection using SNP markers[J]. PLoS One,2009,4(12): e8451
193. Yi G, Lee S K, Hong Y K, et al. Use of Pi5(t) markers in marker-assisted selection to screen forcultivars with resistance to Magnaporthe grisea[J]. Theoretical and applied genetics,2004,109(5):978-985
194. Youens-Clark K, Buckler E, Casstevens T, et al. Gramene database in2010: updates andextensions[J]. Nucleic acids research,2011,39(suppl1): D1085-D1094
195. Yu J, Hu S, Wang J, et al. A draft sequence of the rice genome (Oryza sativa L. ssp. indica)[J].science,2002,296(5565):79-92
196. Yu J, Pressoir G, Briggs W H, et al. A unified mixed-model method for association mapping thataccounts for multiple levels of relatedness[J]. Nature genetics,2006,38(2):203-208
197. Yu J, Wang J, Lin W, et al. The genomes of Oryza sativa: a history of duplications[J]. PLoSbiology,2005,3(2): e38
198. Yu P, Wang C H, Xu Q, et al. Genome-wide copy number variations in Oryza sativa L.[J]. BMCgenomics,2013,14(1):649
199. Yu P, Wang C, Xu Q, et al. Detection of copy number variations in rice using array-basedcomparative genomic hybridization[J]. BMC genomics,2011,12(1):372
200. Zeigler RS, Leong S A, Teng PS. Rice blast disease[M]. Int. Rice Res. Inst.,1994
201. Zhai C, Lin F, Dong Z, et al. The isolation and characterization of Pik, a rice blast resistance genewhich emerged after rice domestication[J]. New phytologist,2011,189(1):321-334
202. Zhang Z, Ersoz E, Lai C Q, et al. Mixed linear model approach adapted for genome-wideassociation studies[J]. Nature genetics,2010,42(4):355-360
203. Zhao K, Aranzana M J, Kim S, et al. An Arabidopsis example of association mapping in structuredsamples[J]. PLoS Genetics,2007,3(1): e4
204. Zhao K, Tung C W, Eizenga G C, et al. Genome-wide association mapping reveals a rich geneticarchitecture of complex traits in Oryza sativa[J]. Nature communications,2011,2:467
205. Zhou B, Qu S, Liu G, et al. The eight amino-acid differences within three leucine-rich repeatsbetween Pi2and Piz-t resistance proteins determine the resistance specificity to Magnaporthegrisea[J]. Molecular plant-microbe interactions,2006,19(11):1216-1228
206. Zhu C, Gore M, Buckler E S, et al. Status and prospects of association mapping in plants[J]. Theplant genome,2008,1(1):5-20
207. Zhu C, Yu J. Nonmetric multidimensional scaling corrects for population structure in associationmapping with different sample types[J]. Genetics,2009,182(3):875-888
208. Zondervan K T, Cardon L R. The complex interplay among factors that influence allelicassociation[J]. Nature Reviews Genetics,2004,5(2):89-100
209. Zong Y, Chen Z, Innes J B, et al. Fire and flood management of coastal swamp enabled first ricepaddy cultivation in east China[J]. Nature,2007,449(7161):459-462