Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2013, Vol. 39 ›› Issue (10): 1711-1719.doi: 10.3724/SP.J.1006.2013.01711

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

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 Online:2013-10-12 Published:2013-07-31
  • Contact: 浦惠明, E-mail: puhuiming@126.com, Tel: 025-84390370

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)

[1] HU Mao-Long, CHENG Li, GUO Yue, LONG Wei-Hua, GAO Jian-Qin, PU Hui-Ming, ZHANG Jie-Fu, CHEN Song. Development and application of the marker for imidazolinone-resistant gene in Brassica napus [J]. Acta Agronomica Sinica, 2020, 46(10): 1639-1646.
[2] Fang-Quan WANG, Jie YANG, Fang-Jun FAN, Wen-Qi LI, Jun WANG, Yang XU, Jin-Yan ZHU, Yun-Yan FEI, Wei-Gong ZHONG. Development and Application of the Functional Marker for Imidazolinone Herbicides Resistant ALS Gene in Rice [J]. Acta Agronomica Sinica, 2018, 44(03): 324-331.
[3] HU Mao-Long, PU Hui-Ming, LONG Wei-Hua, GAO Jian-Qin, QI Cun-Kou, ZHANG Jie-Fu, CHEN Song. Enzymatic Characteristics of Acetolactate Synthase Mutant S638N in Brassica napus and Its Resistance to ALS Inhibitor Herbicides [J]. Acta Agron Sin, 2015, 41(09): 1353-1360.
[4] ZHANG Ya-Jie,LI Jing,PENG Hong-Kun,CHEN Xiu-Bin,ZHENG Hong-Yu,CHEN Sheng-Bei,LIU An-Guo,HU Li-Yong. Dynamic Simulation Model for Growth Duration of Rapeseed (Brassica napus) [J]. Acta Agron Sin, 2015, 41(05): 766-777.
[5] ZHANG Wei-Xin,CAO Hong-Xin,ZHU Yan,LIU Yan,ZHANG Wen-Yu,CHEN Yu-Li,FU Kun-Ya. Morphological Structure Model of Leaf Space Based on Biomass at Pre-Overwintering Stage in Rapeseed (Brassica napus L.) Plant [J]. Acta Agron Sin, 2015, 41(02): 318-328.
[6] ZUO Qing-Song,YANG Hai-Yan,LENG Suo-Hu,CAO Shi,ZENG Jiang-Xue,WU Jiang-Sheng,ZHOU Guang-Sheng. Effects of Nitrogen Fertilizer on Nitrogen Accumulation, Translocation and Nitrogen Use Efficiency in Rapeseed (Brassica napus L.) [J]. Acta Agron Sin, 2014, 40(03): 511-518.
[7] XIE Xiao-Yu,ZHANG Bing,ZHANG Xia,MA Zhong-Lian,LI Jia-Na. Construction and Analysis of SSH Library in Rapeseed (Brassica napus L.) under Drought Stress [J]. Acta Agron Sin, 2013, 39(04): 744-752.
[8] CHEN Guang-Yao;WANG Guo-Huai;LUO Feng;NIE Ming-Jian. Effects of Endogenous Hormones on Pre-harvest Sprouting in Siliqua of Rapeseed (Brassica napus L.) [J]. Acta Agron Sin, 2007, 33(08): 1324-1328.
[9] ZHANG Zi-Long, TAO Shi-Heng, LI Jia-Na. A Comparative Study on Physiological Characteristics during Seedling Stage in Yellow-Seeded and Black-Seeded Rapeseed (Brassica napus L.) [J]. Acta Agronomica Sinica, 2007, 33(05): 837-842.
[10] YU Cheng-Yu; HU Sheng-Wu; ZHANG Chun-Hong; YU Yan-Jun; HE Pei-Ru. The Effect of Chemical Hybridizing Agent EXP on Oilseed Rape [J]. Acta Agron Sin, 2005, 31(11): 1455-1459.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!