Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (09): 1565-1571.doi: 10.3724/SP.J.1006.2014.01565


Establishment and Application of Multiplex PCR System for Two Blast Resistance Genes Pi-ta and Pi-b in Rice

YAO Shu,LIU Yan-Qing,ZHANG Ya-Dong,ZHU Zhen,CHEN Tao,ZHAO Qing-Yong,ZHOU Li-Hui,ZHAO Chun-Fang,YU Xin,WANG Cai-Lin*   

  1. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Jiangsu High Quality Rice R&D Center / Nanjing Branch of China National Center for Rice Improvement, Nanjing 210014, China
  • Received:2014-02-07 Revised:2014-06-16 Online:2014-09-12 Published:2014-07-09
  • Contact: 王才林, E-mail: clwang@jaas.ac.cn, Tel: 025-84390307


Rice blast is one of the important diseases in major rice producing areas of our country. The main blast resistance genes Pi-ta and Pi-b showed good resistance spectrum to rice blast in many rice growing areas of China, and has been utilized in rice breeding and commercial production. In this study, two sets of multiple PCR system for the two rice blast genes were established by selecting the functional markers of two resistance genes of Pi-ta and Pi-b and two susceptible genes of pi-ta and pi-b, respectively, based on the verification of the genotype of the two rice blast genes known in 22 rice varieties. System I could detect the two resistance genes of Pi-ta and Pi-b, while system II could detect the two susceptible genes of pi-ta and pi-b. Genotypes of the two rice blast genes in 336 new japonica lines were detected by using the two sets of multiple PCR system. The results were highly consistent with those tested by the conventional single mark detection method, indicating that the two multiplex PCR systems were stable, reliable and time-saving. They may serve as a rapid and efficient method to identify and screen rice germplasm resources and be applied in marker assistant selection for pyramiding multiple genes (QTLs) of rice blast resistance in rice breeding.

Key words: Rice blast, Molecular marker, Resistance gene, Multiplex PCR system

[1]郑钊, 陈由强, 张建福, 谢华安. 水稻稻瘟病抗性基因的定位、克隆及应用. 分子植物育种, 2009, 7: 385–392

Zheng Z, Chen Y Q, Zhang J F, Xie H A. Mapping cloning of rice blast resistance genes and their application. Mol Plant Breed, 2009, 7: 385–392 (in Chinese with English abstract)

[2]沈瑛, 朱培良, 袁筱萍, Levy M, Decker M, Talbot N, Hamer J E. 中国稻瘟病菌的遗传多样性. 植物病理学报, 1993, 23: 309−313

Shen Y, Zhu P L, Yuan X P, Levy M, Decker M, Talbot N, Hamer J E. Genetic diversity of Magnaporthe grisea in China. Acta Phytopathol Sin, 1993, 23: 309−313 (in Chinese)

[3]Hittalmani S, Parco A, Mew T V, Zeigler R S, Huang N. Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theor Appl Genet, 2000, 100: 1121–1128

[4]Zhai C, Lin F, Dong Z Q, He X Y, Yuan B, Zeng X S, Wang L, Pan Q H. The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication. New Phytologist, 2011, 189: 321−334

[5]Hayashi N, Inoue H, Kato T, Shirota M, Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T, Yano M, Takatsuji H. Durable panicle blast resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication. Plant J, 2010, 64: 498–510

[6]Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M, Saitoh H, Fujibe T, Matsumura H, Shenton M. Galam D C, Undan J, Ito A, Sone T, Terauchi R. A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes. Plant J, 2011, 66: 467–479

[7]Yuan B, Zhai C, Wang W J, Zeng X S, Xu X K, Hu H Q, Lin F, Wang L, Pan Q H. The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes. Theor Appl Genet, 20l1, 122: 1017–1028

[8]Takahashi A, Hayashi N, Miyao A, Hirochike H. Unique features of the rice blast resistance Pish locus revealed by large scale ret retrotransposon-tagging. BMC Plant Biol, 2010, 10: 175

[9]Wang Z X, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T. The Pi-b gene for rice blast resistance belongs to the nucleotide binding and leucine rich repeat class of plant resistance genes. Plant J, 1999, 19: 55–64

[10]Bryan G T, Wu K S, Farrall L, Jia Y L, Hershey H P, Mcadams S A, Fault K N, Donaldson G K, Renato T, Valent B. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell, 2000, 12: 2033–2045

[11]Wang Z, Jia Y, Rutger J N, Xia Y, Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene. Plant Breed, 2007, 126: 36–42

[12]刘洋, 徐培洲, 张红宇, 徐建第, 吴发强, 吴先军. 水稻抗稻瘟病Pi-b基因的分子标记辅助选择与应用. 中国农业科学, 2008, 41: 9–14

Liu Y, Xu P Z, Zhang H Y, Xu J D, Wu F Q, Wu X J. Marker-assisted selection and application of blast resistant gene Pib in rice. Sci Agric Sin, 2008, 41: 9–14 (in Chinese with English abstract)

[13]李进斌, 王甜, 许明辉. 云南地方稻种抗稻瘟病基因Pi-ta和Pi-b的鉴定. 中国水稻科学, 2012, 26: 593–599

Li J B, Wang T, Xu M H. Identification 0f Pi-ta and Pi-b genes for rice blast resistance of rice landraces from Yunnan Province. Chin J Rice Sci, 2012, 26: 593–599 (in Chinese with English abstract)

[14]刘华招, 刘延, 刘化龙, 徐正进, 陈温福. 黑龙江省种植品种中稻瘟病抗性基因Pi-b和Pi-ta的分布. 东北农业大学学报, 2011, 42(1): 27−31

Liu H Z, Liu Y, Liu H L, Xu Z J, Chen W F. Distribution of two blast resistance genes Pib and Pita in major rice cu1tivars in Heilongjiang Province in China. J. Northeast Agric Univ, 2011, 42(1): 27−31 (in Chinese with English abstract)

[15]时克, 雷财林, 程治军, 许兴涛, 王久林, 万建民. 稻瘟病抗性基因Pi-ta和Pi-b在我国水稻主栽品种中的分布. 植物遗传资源学报, 2009, 10: 21–26

Shi K, Lei C L, Cheng Z J, Xu X T, Wang J L, Wan J M. Distribution of two blast resistance genes Pita and Pi-b in major rice cultivars in China. J Plant Genet Resour, 2009, 10: 21−26 (in Chinese with English abstract)

[16]Ma W, Zhang W, Gale K R. Multiplex-PCR typing of high molecular weight glutenin alleles in wheat. Euphytica, 2003, 134: 51–60

[17]Nakamura T, Vrinten P, Saito M, Konda M. Rapid classification of partial waxy wheat using PCR-based markers. Genome, 2002, 45: l150–l156

[18]戴小军, 杨远柱, 周亮, 梁满中, 胡小淳, 陈良碧. 抗稻瘟病水稻资源抗性基因Pi-ta、Pi-b、Pi9以及Pikm的分布研究. 生命科学研究, 2012, 16: 340−344

Dai X J, Yang Y Z, Zhou L, Liang M Z, Hu X C, Chen L B. Distribution research of blast resistance genes Pi-ta, Pi-b, Pi-9 and Pikm in blast-resistant rice resources. Life Sci Res, 2012, 16: 340−344 (in Chinese with English abstract)

[19]Li W, Lei C L, Cheng Z J, Jia Y L, Huang D Y, Wang J L, Wang J K, Zhang X, Su N, Guo X P, Zhai H Q, Wan J M. Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding. Mol Breed, 2008, 22: 141–149

[20]王忠华, 贾育林, 吴殿星, 夏英武. 水稻抗稻瘟病基因Pi-ta的分子标记辅助选择. 作物学报, 2004, 30: 1259–1265

Wang Z H, Jia Y L, Wu D X, Xia Y W, Molecular marker-assisted selection of the rice blast resistance gene Pi-ta. Acta Agron Sin, 2004, 30: 1259–1265 (in Chinese with English abstract)

[21]Fjellstrom R, Conaway-Bormans C A, McClung A M, Marchetti M A, Shank A R, Park W D. Development of DNA markers suitable for marker assisted selection of three Pi genes conferring resistance to multiple Pyricularia grlsea pathotypes. Crop Sci, 2004, 44: 1790–1798

[22]杨杰, 杨金欢, 王军, 范方军, 朱金燕, 曹卿, 田胜尼, 仲维功. 稻瘟病抗病基因Pi-ta和Pi-b在中国水稻地方品种中的分布. 华北农学报, 2011, 26(3): 1–6

Yang J, Yang J H, Wang J, Fan F J, Zhu J Y, Cao Q, Tian S N, Zhong W G. Distribution of two blast resistant genes Pita and Pib in landrace rice in China. Acta Agric Boreali-Sin, 2011, 26(3): 1–6 (in Chinese with English abstract)

[23]李进斌, 李成云, 陈艳, 雷财林, 凌忠专. 二十二个抗稻瘟病基因在云南的利用价值评价. 植物保护学报, 2005, 6: 113–119

Li J B, Li C Y, Chen Y, Lei C L, Ling Z Z. Utilization value of twenty-two blast resistance genes in Yunnan. Acta Phytophy Sin, 2005, 6: 113–119 (in Chinese with English abstract)

[24]王国珍, 茹庆华, 高立原, 李华. 日本水稻抗瘟基因对宁夏稻瘟病菌的抗性研究.西北农业学报, 1997, 6(3): 1–4

Wang G Z, Ru Q H, Gao L Y, Li H. Studies on genes for resistance blast fungus in Ningxia region. Acta Agric Bori-Occide Sin, 1997, 6(3): 1–4 (in Chinese with English abstract)

[25]陆凡, 陈志谊, 刘永锋, 郑小波. 江苏省稻瘟病菌毒性的群体结构分析. 植物保护学报, 2002, 29: 289–294

Lu F, Chen Z Y, Liu Y F, Zheng X B. Analysis of population virulences of Magnaporthe grisea in Jiangsu Province. Acta Phytophy Sin, 2002, 29: 289–294 (in Chinese with English abstract)

[26]任金平, 郭晓莉, 郑民, 杨晓辉, 刘晓梅, 李莉, 孙辉. 吉林省稻瘟病菌对水稻品种的致病谱研究. 吉林农业科学, 2006, 31(6): 35–37

Ren J P, Guo X L, Zheng M, Yang X H, Liu X M, Li L, Sun H. Studies on pathogenic spectrum of rice varieties with Magnaporth grisea in Jilin Province. J Jilin Agric Sci, 2006, 31(6): 35–37 (in Chinese with English abstract)

[27]Liu Z B, Gao Q R, Wang R X, Qiao X L, Qiu X M. Application of multiplex PCR to studies on plant biology. Mol Plant Breed, 2005, 3: 261–268

[1] DENG Zhao, JIANG Nan, FU Chen-Jian, YAN Tian-Zhe, FU Xing-Xue, HU Xiao-Chun, QIN Peng, LIU Shan-Shan, WANG Kai, YANG Yuan-Zhu. Analysis of blast resistance genes in Longliangyou and Jingliangyou hybrid rice varieties [J]. Acta Agronomica Sinica, 2022, 48(5): 1071-1080.
[2] YANG De-Wei, WANG Xun, ZHENG Xing-Xing, XIANG Xin-Quan, CUI Hai-Tao, LI Sheng-Ping, TANG Ding-Zhong. Functional studies of rice blast resistance related gene OsSAMS1 [J]. Acta Agronomica Sinica, 2022, 48(5): 1119-1128.
[3] ZHU Zheng, WANG Tian-Xing-Zi, CHEN Yue, LIU Yu-Qing, YAN Gao-Wei, XU Shan, MA Jin-Jiao, DOU Shi-Juan, LI Li-Yun, LIU Guo-Zhen. Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae [J]. Acta Agronomica Sinica, 2022, 48(5): 1129-1140.
[4] FU Mei-Yu, XIONG Hong-Chun, ZHOU Chun-Yun, GUO Hui-Jun, XIE Yong-Dun, ZHAO Lin-Shu, GU Jia-Yu, ZHAO Shi-Rong, DING Yu-Ping, XU Yan-Hao, LIU Lu-Xiang. Genetic analysis of wheat dwarf mutant je0098 and molecular mapping of dwarfing gene [J]. Acta Agronomica Sinica, 2022, 48(3): 580-589.
[5] MA Hong-Bo, LIU Dong-Tao, FENG Guo-Hua, WANG Jing, ZHU Xue-Cheng, ZHANG Hui-Yun, LIU Jing, LIU Li-Wei, YI Yuan. Application of Fhb1 gene in wheat breeding programs for the Yellow-Huai Rivers valley winter wheat zone of China [J]. Acta Agronomica Sinica, 2022, 48(3): 747-758.
[6] WANG Yin, FENG Zhi-Wei, GE Chuan, ZHAO Jia-Jia, QIAO Ling, WU Bang-Bang, YAN Su-Xian, ZHENG Jun, ZHENG Xing-Wei. Identification of seedling resistance to stripe rust in wheat-Thinopyrum intermedium translocation line and its potential application in breeding [J]. Acta Agronomica Sinica, 2021, 47(8): 1511-1521.
[7] HE Jun-Yu, YIN Shun-Qiong, CHEN Yun-Qiong, XIONG Jing-Lei, WANG Wei-Bin, ZHOU Hong-Bin, CHEN Mei, WANG Meng-Yue, CHEN Sheng-Wei. Identification of wheat dwarf mutants and analysis on association between the mutant traits of the dwarf plants [J]. Acta Agronomica Sinica, 2021, 47(5): 974-982.
[8] WANG Heng-Bo, CHEN Shu-Qi, GUO Jin-Long, QUE You-Xiong. Molecular detection of G1 marker for orange rust resistance and analysis of candidate resistance WAK gene in sugarcane [J]. Acta Agronomica Sinica, 2021, 47(4): 577-586.
[9] JIANG Wei, PAN Zhe-Chao, BAO Li-Xian, ZHOU Fu-Xian, LI Yan-Shan, SUI Qi-Jun, LI Xian-Ping. Genome-wide association analysis for late blight resistance of potato resources [J]. Acta Agronomica Sinica, 2021, 47(2): 245-261.
[10] ZHANG Xue-Cui, SUN Su-Li, LU Wei-Guo, LI Hai-Chao, JIA Yan-Yan, DUAN Can-Xing, ZHU Zhen-Dong. Identification of resistance gene against phytophthora root rot in new soybean lines breeded in Henan province [J]. Acta Agronomica Sinica, 2021, 47(2): 275-284.
[11] ZHANG Rong-Yue, WANG Xiao-Yan, YANG Kun, SHAN Hong-Li, CANG Xiao-Yan, LI Jie, WANG Chang-Mi, YIN Jiong, LUO Zhi-Ming, LI Wen-Feng, HUANG Ying-Kun. Identification of brown rust resistance and molecular detection of Bru1 gene in new and main cultivated sugarcane varieties [J]. Acta Agronomica Sinica, 2021, 47(2): 376-382.
[12] ZHANG Huan, LUO Huai-Yong, LI Wei-Tao, GUO Jian-Bin, CHEN Wei-Gang, ZHOU Xiao-Jing, HUANG Li, LIU Nian, YAN Li-Ying, LEI Yong, LIAO Bo-Shou, JIANG Hui-Fang. Genome-wide identification of peanut resistance genes and their response to Ralstonia solanacearum infection [J]. Acta Agronomica Sinica, 2021, 47(12): 2314-2323.
[13] GUO Qing-Qing, ZHOU Rong, CHEN Xue, CHEN Lei, LI Jia-Na, WANG Rui. Location and InDel markers for candidate interval of the orange petal gene in Brassica napus L. by next generation sequencing [J]. Acta Agronomica Sinica, 2021, 47(11): 2163-2172.
[14] HUANG Yi-Wen, DAI Xu-Ran, LIU Hong-Wei, YANG Li, MAI Chun-Yan, YU Li-Qiang, YU Guang-Jun, ZHANG Hong-Jun, LI Hong-Jie, ZHOU Yang. Relationship between the allelic variations at the Ppo-A1 and Ppo-D1 loci and pre-harvest sprouting resistance in wheat [J]. Acta Agronomica Sinica, 2021, 47(11): 2080-2090.
[15] GUO Yan-Chun, ZHANG Li-Lan, CHEN Si-Yuan, QI Jian-Min, FANG Ping-Ping, TAO Ai-Fen, ZHANG Lie-Mei, ZHANG Li-Wu. Establishment of DNA molecular fingerprint of applied core germplasm in jute (Corchorus spp.) [J]. Acta Agronomica Sinica, 2021, 47(1): 80-93.
Full text



No Suggested Reading articles found!