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作物学报 ›› 2013, Vol. 39 ›› Issue (10): 1711-1719.doi: 10.3724/SP.J.1006.2013.01711

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用

胡茂龙,龙卫华,高建芹,付三雄,陈锋,周晓婴,彭琦,张维,浦惠明*,戚存扣,张洁夫,陈松   

  1. 江苏省农业科学院经济作物研究所 / 国家油料作物改良中心南京分中心 / 农业部长江下游棉花与油菜重点实验室，南京 210014
  • 收稿日期:2013-02-26 修回日期:2013-05-25 出版日期:2013-10-12 网络出版日期:2013-07-31
  • 通讯作者: 浦惠明, E-mail: puhuiming@126.com, Tel: 025-84390370
  • 基金资助:

    本研究由国家自然科学基金项目(311101174)项目, 国家高技术研究发展计划(863计划)项目(2011AA10A10403), 江苏省自然科学基金项目(BK2011679), 江苏省农业自主创新基金[cx(11)4012]和南京农业大学作物遗传与种质创新国家重点实验室开放基金(ZW2011006)资助。

Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant Gene BnALS1R in Brassica napus

HU Mao-Long,LONG Wei-Hua,GAO Jian-Qin,FU San-Xiong,CHEN Feng,ZHOU Xiao-Yin,PENG Qi,ZHANG Wei,PU Hui-Ming*,QI Cun-Kou,ZHANG Jie-Fu,CHEN Song   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Sub-center, National Center of Oil Crops Improvement / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture, Nanjing 210014, China
  • Received:2013-02-26 Revised:2013-05-25 Published:2013-10-12 Published online:2013-07-31
  • Contact: 浦惠明, E-mail: puhuiming@126.com, Tel: 025-84390370

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摘要：

油菜抗咪唑啉酮类除草剂基因BnALS1R是从抗性突变体M9中克隆获得，抗性基因BnALS1R与野生型基因BnALS1存在1处SNP，即乙酰乳酸合酶第638位丝氨酸残基被天冬酰胺酸替代。为获得油菜抗除草剂基因BnALS1R的分子标记，根据该处点突变，结合获得的BnALS3与BnALS1序列，开发30条等位基因特异PCR (allele-specific PCR，AS-PCR)引物，采用筛选出的3条AS-PCR引物在F2、BC1和BC2群体中进行PCR扩增。结果表明，该标记有效检测出群体中存在的3种基因型，其分离比分别为1∶2∶1、1∶1、1∶1，均遵循单基因遗传规律。应用该标记对获得的抗性恢复系进行PCR扩增，结果发现所有抗性恢复系均能扩增出抗性基因BnALS1R目的条带，表明3条标记引物可应用于抗性基因的检测。AS-PCR标记的获得将促进以抗性基因进行油菜抗除草剂分子标记辅助选择育种。

关键词: 油菜, 咪唑啉酮类除草剂, BnALS1R, 乙酰乳酸合成酶, 等位基因特异PCR

Abstract:

A BnALS1R gene conferring resistance to the imidazolinone herbicides was previously isolated from an ALS (acetolactate synthase) inhabiting herbicide-resistant mutant line M9 in Brassica napus. A single nucleotide polymorphism leading to an amino acid substitution from serine to asparagine at site 638 of ALS was found between the BnALS1R in the mutant and BnALS1 in the wild type. We reported here the development of polymorphic molecular marker for allele-specific PCR (AS-PCR) assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes. Thirty primers were designed according to the SNP (site 638, Ser was replaced by Asn) and the difference in DNA sequence between the BnALS1 and BnALS3 cloned from M9 and other susceptible cultivars. Three primers of them with polymorphism were obtained, which can differentiate the homozygous resistant M9 from susceptible rapeseed. Meanwhile, these PCR markers can effectively discriminate among three genotypes using polymorphic primers and were evaluated in F2, BC1, and BC2 populations. Observed segregation ratios fitted the expected 1:2:1, 1:1, and 1:1 ratios, respectively, which confirms the single-locus Mendel model. Furthermore, we used the markers for detecting the resistant gene and evaluating the size of DNA segments introgressed from the M9 donor in the resistant restoring lines containing BnALS1R. All the resistant restoring lines had the DNA banding pattern of BnALS1R. Therefore, PCR-based assays using the markers could be used for the detection and characterization of the herbicide-resistant gene BnALS1R in rapeseed. The validated AS-PCR markers will facilitate the breeding of herbicide-resistant rapeseed by using BnALS1R in marker-assisted selection.

Key words: Rapeseed (Brassica napus L.), Imidazolinone herbicides, BnALS1R, Acetolactate synthase, Allele-Specific PCR

[1]Tan S, Richard R E, Mark L D, Bijay K S, Dale L S. Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci, 2005, 61: 246–257



[2]Jennifer A M, Pang S S, Jack K S, Luke W G, Ronald G D. Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase. Proc Natl Acad Sci USA, 2006, 103: 569–573



[3]Ronald G D, Jennifer A M, Luke W G. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol Bioch, 2008, 46: 309–324



[4]Lee H, Sachin R, Neeraj K, Burkea I, Yenisha J P, Gilla K S, Wettsteina D V, Steven E U. Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides. Proc Natl Acad Sci USA, 2011, 108: 8909–8913



[5]Carlos A S, Mariano B, Mariel E, Sherry R, Ascenzi W R. Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower. Theor Appl Genet, 2008, 118: 105–112



[6]Carlos A S, Mariano B. Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sun?ower. Theor Appl Genet, 2012, 124: 355–364



[7]Carlos A S, Mariano B, Emiliano A, Brigitte W. Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the Ahasl1 locus of sun?ower. Theor Appl Genet, 2012, 124: 385–396



[8]Hattori J, Brown D, Mourad G, Labbe H, Ouellet T, Sunohara G, Rutledge R, King J, Miki B. An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance. Mol Gen Genet, 1995, 246: 419–425



[9]Swanson E B, Herrgesell M J, Arnoldo M, Sippell D W, Wong R S C. Microspore mutagenesis and selection: canola plants with field tolerance to the imidazolinones. Theor Appl Genet, 1989, 78: 525–530



[10]Gao J-Q(高建芹), Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Long W-H(龙卫华), Hu M-L(胡茂龙), Chen S(陈松), Chen X-J(陈新军), Chen F(陈锋), Gu H(顾慧). Identification of imidazolidone-resistant oilseed rape mutant. J Plant Genet Resour (植物遗传资源学报), 2010, 11(3): 369–373 (in Chinese with English abstract)



[11]Gao J-Q(高建芹), Pu H-M(浦惠明), Long W-H(龙卫华), Hu M-L(胡茂龙), Qi C-K(戚存扣), Zhang J-F(张洁夫). Resistance of imidzolidone--resistant oilseed rape to nonselective Herbicide imazethapyr. Jiangsu J Agric Sci (江苏农业学报), 2010, 26(6): 1186–1191 (in Chinese with English abstract)



[12]Hu M-L(胡茂龙), Pu H-M(浦惠明), Gao J-Q(高建芹), Long W-H(龙卫华), Qi C-K(戚存扣), Zhang J-F(张洁夫), Chen S(陈松). Inheritance and gene cloning of an ALS inhabiting herbicide-resistant mutant line M9 in Brassica napus. Sci Agri Sin (中国农业科学), 2012, 45(20): 4326–4334 (in Chinese with English abstract)



[13]Drenkard E, Richter B G, Rozen S, Stutius L M, Angell N A, Mindrinos M, Cho R J, Oefner P J, Davis R W, Ausubel F M. A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiol, 2000, 124: 1483–1492



[14]Kadaru S, Zhang W Q, Yadav A, James H O. Development and application of allele-specific PCR assays for imazethapyr resistance in rice (Oryza sativa). Euphytica, 2008, 160: 431–438



[15]Pu H-M(浦惠明), Gao J-Q(高建芹), Long W-H(龙卫华), Hu M-L(胡茂龙), Zhang J-F(张洁夫), Chen S(陈松), Chen X-J(陈新军), Chen F(陈锋), Gu H(顾慧), Fu S-X(付三雄), Qi C-K(戚存扣). Studies on inheritance of imidazolinones resistance in Brassica napus and its utilization. Chin J Oil Crop Sci (中国油料作物学报), 2011, 33(1): 15–19 (in Chinese with English abstract)



[16]Hu M-L(胡茂龙), Long W-H(龙卫华), Gao J-Q(高建芹), Chen X-J(陈新军), Zhang J-F(张洁夫), Chen S(陈松) Qi C-K(戚存扣), Pu H-M(浦惠明). Cloning of glutathione peroxidase BnGPX1 gene from Brassica napus L. and its expression under abiotic stresses. Chin J Oil Crop Sci (中国油料作物学报), 2011, 33(4): 331–337 (in Chinese with English abstract)



[17]Rutledge R G, Quellet T, Hattori J, Miki B L. Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family. Mol Gen Genet, 1991, 229: 31–40



[18]Hu M-L(胡茂龙), Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Long W-H(龙卫华), Chen S(陈松), Chen X-J(陈新军), Chen F(陈锋), Gu H(顾慧). Gene Cloning and Application of an ALS Inhabiting Herbicide-Resistant Mutant Line M9 in Brassica napus. China Patent (CN201010232607.4), 2012



[19]Konieczny A, Ausubel F. A procedure for mapping Arabidopsis mutations using co-dominant ecotype specific PCR-based markers. Plant J, 1993, 4: 403–410



[20]Michaels S D, Amasino R M. A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J, 1998, 14: 381–385



[21]Neff M M, Neff J D, Chory J, Pepper A E. dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J, 1998, 14: 387–392



[22]Chen X, Martin T, Crystal L S, Aliaa E M, Saleh S, Weselake R J. Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus. Plant Physiol, 2011, 155: 851–865



[23]Ouellet T, Rutledget R G, Miki B L. Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression. Plant J, 1992, 2: 321–330



[24]Wei B(卫波), Jing R-L(景蕊莲), Wang C-S(王成社), Chang X-P(昌小平). Assaying single nucleotide polymorphism in wheat (Triticum aestivum L.) with allele-specific PCR. Sci Agri Sin (中国农业科学), 2006, 39(7): 1313–1320 (in Chinese with English abstract)



[25]Hayashi K, Hashimoto N, Daigen M, Ashikawa I. Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet, 2004, 108: 1212–1220



[26]Chen J-B(陈吉宝), Jing R-L(景蕊莲), Yuan H-Y(员海燕), Wei B(卫波). Development of allele-specific PCR. J Plant Genet Resour (植物遗传资源学报), 2005, 6(4): 469–47 (in Chinese with English abstract)



[27]Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Fu S-Z(傅寿仲), Chen X-J(陈新军), Chen S(陈松), Zhao X-X(赵祥祥). Studies on the gene flow from herbicide-tolerant GM rapeseed to its close relative crops. Acta Ecol Sin (生态学报), 2005, 25(3): 196–203 (in Chinese with English abstract)



[28]Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Fu S-Z(傅寿仲), Chen X-J(陈新军), Chen S(陈松), Zhao X-X(赵祥祥). The studies on gene flow from GM herbicide-tolerant rapeseed to cruciferous weeds. Acta Ecol Sin (生态学报), 2005, 25(4): 910–916 (in Chinese with English abstract)
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