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

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

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。

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.