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

doi:10.3724/SP.J.1006.2013.01711

Abstract

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 BC₂populations. 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

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. Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant Gene BnALS1R in Brassica napus[J].Acta Agron Sin, 2013, 39(10): 1711-1719.

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

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