Data from: Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): Insights into resistance evolution in a hexaploid species

Fig. 5. Plastidic ACCase gene partial sequence alignment: Partial sequence alignment (from cloned fragments) showing the three haplotype sequences of the 2088, 1781 and 2078 mutations identified in the present study, corresponding respectively to the ACCase gene copies Acc1;1, Acc1;2, and Acc1;3 in wild oat (Christoffers et al., 2002). Note that the sequence region containing the 1781, 2078 or 2088 codon was not included due to infrequent informative SNPs. Boxed regions indicate SNPs discriminating the three haplotypes (GenBank accession AF231335 for Acc1;1-1, AF231334 for Acc1;1-2, AF231336 for Acc1;2 and AF464875 for Acc1;3).

Fig. S4( d)CAPS analysis of clones: Fig. S4 (d)CAPS marker analysis of ACCase mutations at (a) 1781, (b) 2078 or (c) 2088 in representative E. coli clones containing the 1122 kb ACCase cDNA insert. Each lane represents a single clone analysed by each of the three markers. Note that none of these clones contain more than one mutation (R).

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.

图5 质体乙酰辅酶A羧化酶（plastidic acetyl-coenzyme A carboxylase，ACCase）基因部分序列比对：本研究中鉴定得到的2088、1781及2078位点突变的三种单倍型序列的部分比对结果（源自克隆片段），分别对应野燕麦中ACCase基因拷贝Acc1;1、Acc1;2与Acc1;3（Christoffers等，2002）。需注意，由于信息性单核苷酸多态性（single nucleotide polymorphism，SNP）较为稀少，包含1781、2078或2088密码子的序列区域未纳入本次比对。框选区域为区分三种单倍型的单核苷酸多态性位点（Acc1;1-1的GenBank登录号为AF231335，Acc1;1-2为AF231334，Acc1;2为AF231336，Acc1;3为AF464875）。

补充图4(d) 克隆的酶切扩增多态性序列（cleaved amplified polymorphic sequence，CAPS）分析：针对携带1122 kb ACCase互补DNA（complementary DNA，cDNA）插入片段的代表性大肠杆菌（Escherichia coli，E. coli）克隆，分别对1781(a)、2078(b)及2088(c)位点的ACCase突变开展CAPS标记分析。每个泳道代表通过三种标记之一检测的单个克隆。需注意，所有受试克隆均未携带超过一种突变（R）。

许多抗除草剂杂草物种为多倍体，但目前学界对多倍体中抗性突变的演化机制仍知之甚少。六倍体野燕麦（Avena fatua）是全球性农田恶性杂草，诸多种群已演化出除草剂抗性。本研究针对六倍体野燕麦中的质体乙酰辅酶A羧化酶（ACCase）抑制型除草剂抗性展开系统研究，结果显示抗性个体可表达一至三种不同的质体ACCase基因抗性突变：Ile-1781-Leu、Asp-2078-Gly及Cys-2088-Arg。
以ACCase抗性突变作为分子标记，结合遗传、分子与生化研究手段，我们在单个抗性野燕麦植株中获得三项关键发现：(1) 至多三个不连锁的ACCase基因座按照二体遗传的孟德尔定律独立分离；(2) 这三个同源ACCase基因均发生转录，且各自可携带独立的抗性突变；(3) 在六倍体遗传背景下，单个ACCase抗性突变仅赋予相对低水平的除草剂抗性，这与禾本科（Poaceae）非相关二倍体杂草物种中同一突变所介导的高水平抗性形成显著差异。单个ACCase抗性突变所带来的低抗性水平，可能源于六倍体野燕麦中同源等位基因表达的易感型ACCase产生的稀释效应，以及/或同源ACCase基因拷贝的差异表达。因此，六倍体野燕麦的多倍体特性可能延缓除草剂抗性的演化进程。
本研究同时发现野燕麦种群中存在非靶标抗性机制共存的现象。综上，上述结果表明，六倍体野燕麦的除草剂抗性及其演化过程，较非相关二倍体禾本科杂草更为复杂。本研究数据为解析多倍体杂草抗性演化这一极具挑战性的研究任务提供了重要的起始基础。