Kaundun Shiv S, Cleere Sally M, Stanger Carole P, Burbidge Judith M, Windass John D
Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, UK.
Pest Manag Sci. 2006 Nov;62(11):1082-91. doi: 10.1002/ps.1283.
The I1781L amino acid substitution in the target ACCase enzyme causes broad resistance to ACCase inhibitor herbicides in several monocotyledenous weeds of agronomic importance. This mutation results from a substitution of an adenine (A) residue by either a thymine (T) or cytosine (C) at position 5341 in Alopecurus myosuroides Huds and at an equivalent position in Lolium species, Avena fatua L. and Setaria viridis (L.) Beauv. Two different procedures, the PCR-based allele-specific assay (ASA) and the derived cleaved amplified polymorphic sequence (dCAPS) method, have previously been described for detecting this mutation. These methods are, however, only amenable to low sample throughput and are used in the analysis of single plants. Here, an alternative high-throughput ARMS/Scorpion real-time quantitative PCR (Q-PCR) method for measuring levels of the I1781L mutation in pools of leaf and seed samples of Lolium populations is presented. The limit of detection for C and T mutant alleles in a background of wild-type A is 0.02 and 0.0003% respectively. In this study, DNA from batches of 24 leaf segments measuring 0.5 cm from different plants or 1000 seeds could be conveniently extracted and accurately analysed. As part of assay validation, the comparative analysis of five geographically distinct Lolium populations with dCAPS and Q-PCR procedures demonstrated the accuracy of the latter method, and the three possible II1781, IL1781 and LL1781 ACCase genotypes being distributed as predicted by the Hardy-Weinberg principle. Given the dominance of the L1781 over the I1781 allele at recommended field rates for most ACCase inhibitors, the frequency of herbicide survivors in the field owing only to the presence of the I1781L mutation is thus predicted to be 2pq + q(2), where p and q are the frequencies of the I1781 and L1781 alleles as determined by Q-PCR. The Q-PCR assay established allows detection of very low levels of the L1781 ACCase mutation before resistance would normally be discernible in the field. Therefore, it offers the opportunity to tackle resistance at its very onset, potentially avoiding implementation of complicated and often costly weed management practices.
靶标乙酰辅酶A羧化酶（ACCase）中的I1781L氨基酸替换导致几种具有重要农艺价值的单子叶杂草对ACCase抑制剂类除草剂产生广泛抗性。这种突变是由于在鼠尾看麦娘（Alopecurus myosuroides Huds）的第5341位以及黑麦草属物种、野燕麦（Avena fatua L.）和绿色狗尾草（Setaria viridis (L.) Beauv.）的等效位置上，腺嘌呤（A）残基被胸腺嘧啶（T）或胞嘧啶（C）取代所致。此前已描述了两种不同的方法，即基于聚合酶链反应（PCR）的等位基因特异性分析（ASA）和衍生的酶切扩增多态性序列（dCAPS）方法来检测这种突变。然而，这些方法仅适用于低样本通量，且用于单株植物的分析。在此，我们提出了一种替代的高通量扩增不应性突变系统/蝎形实时定量聚合酶链反应（Q-PCR）方法，用于测量黑麦草种群叶片和种子样本库中I1781L突变的水平。在野生型A背景下，C和T突变等位基因的检测限分别为0.02%和0.0003%。在本研究中，可以方便地提取来自不同植株的24个0.5厘米长叶片切段批次或1000粒种子的DNA，并进行准确分析。作为分析验证的一部分，采用dCAPS和Q-PCR方法对五个地理上不同的黑麦草种群进行比较分析，证明了后一种方法的准确性，并且三种可能的II1781、IL1781和LL1781 ACCase基因型的分布符合哈迪-温伯格原理的预测。鉴于在大多数ACCase抑制剂的推荐田间施用量下，L1781等位基因对I1781等位基因呈显性，因此预计仅由于I1781L突变而在田间存活的除草剂抗性植株的频率为2pq + q²，其中p和q是通过Q-PCR确定的I1781和L1781等位基因的频率。所建立的Q-PCR分析方法能够在田间通常可识别抗性之前检测到极低水平的L1781 ACCase突变。因此，它提供了在抗性刚开始出现时就加以应对的机会，有可能避免实施复杂且往往成本高昂的杂草管理措施。
