Rapid adaptation of weeds to herbicide applications in agriculture through resistance development is a widespread phenomenon. In particular, the grass Alopecurus myosuroides is an extremely problematic weed in cereal crops with the potential to manifest resistance in only a few generations. Target-site resistances (TSRs), with their strong phenotypic response, play an important role in this rapid adaptive response. Recently, using PacBio's long-read amplicon sequencing technology in hundreds of individuals, we were able to decipher the genomic context in which TSR mutations occur. However, sequencing individual amplicons are costly and time-consuming, thus impractical to implement for other resistance loci or applications. Alternatively, pool-based approaches overcome these limitations and provide reliable allele frequencies, although at the expense of not preserving haplotype information. In this proof-of-concept study, we sequenced with PacBio High Fidelity (HiFi) reads long-range amplicons (13.2 kb), encompassing the entire ACCase gene in pools of over 100 individuals, and resolved them into haplotypes using the clustering algorithm PacBio amplicon analysis (pbaa), a new application for pools in plants and other organisms. From these amplicon pools, we were able to recover most haplotypes from previously sequenced individuals of the same population. In addition, we analysed new pools from a Germany-wide collection of A. myosuroides populations and found that TSR mutations originating from soft sweeps of independent origin were common. Forward-in-time simulations indicate that TSR haplotypes will persist for decades even at relatively low frequencies and without selection, highlighting the importance of accurate measurement of TSR haplotype prevalence for weed management.

中文翻译：

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通过基于池的扩增子测序实现黑草中靶位点抗性基因座 ACCase 的深度单倍型分析

杂草通过抗性发展而快速适应农业中除草剂的应用是一种普遍现象。特别是，看麦穗草（Alopecurus myosuroides）是谷类作物中一种极其有问题的杂草，仅在几代之内就有可能表现出抗性。靶点抗性（TSR）具有强烈的表型反应，在这种快速适应性反应中发挥着重要作用。最近，通过对数百个个体使用 PacBio 的长读长扩增子测序技术，我们能够破译 TSR 突变发生的基因组背景。然而，对单个扩增子进行测序既昂贵又耗时，因此对于其他抗性基因座或应用来说是不切实际的。或者，基于池的方法克服了这些限制并提供可靠的等位基因频率，尽管代价是不保留单倍型信息。在这项概念验证研究中，我们使用 PacBio 高保真 (HiFi) 读取长程扩增子 (13.2 kb) 进行测序，其中包含 100 多个个体池中的整个ACCase 基因，并使用聚类算法 PacBio 将它们解析为单倍型扩增子分析 ( pbaa )，植物和其他生物体池的新应用。从这些扩增子库中，我们能够从同一群体的先前测序个体中恢复大多数单倍型。此外，我们分析了德国范围内的A. myosuroides种群的新库，发现源自独立起源的软扫描的 TSR 突变很常见。实时模拟表明，即使在相对较低的频率且没有选择的情况下，TSR 单倍型也将持续数十年，这凸显了准确测量 TSR 单倍型流行率对于杂草管理的重要性。