Institute of Plant Breeding, Seed Science and Population Genetics, University of Hohenheim, Stuttgart, Germany.
Department of Molecular Biology, Max Planck Institute for Biology Tübingen, Tübingen, Germany.
Plant Biotechnol J. 2023 Jun;21(6):1240-1253. doi: 10.1111/pbi.14033. Epub 2023 Apr 10.
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.
杂草在农业中通过抗性发展对除草剂的快速适应是一种普遍现象。特别是雀麦属的节节麦是谷物作物中一种极具问题性的杂草,它只需几代时间就可能表现出抗性。靶标位点抗性(TSR)具有强烈的表型反应,在这种快速适应性反应中起着重要作用。最近,我们使用 PacBio 的长读扩增子测序技术对数百个个体进行了研究,从而能够解析 TSR 突变发生的基因组背景。然而,单个扩增子的测序既昂贵又耗时,因此对于其他抗性基因座或应用来说不切实际。相反,池化方法克服了这些限制,提供了可靠的等位基因频率,尽管是以牺牲单倍型信息为代价的。在这项概念验证研究中,我们使用 PacBio High Fidelity(HiFi)读取长距离扩增子(13.2 kb)对超过 100 个个体的池进行了测序,并使用聚类算法 PacBio amplicon analysis(pbaa)将其解析为单倍型,这是植物和其他生物体中池化的一种新应用。从这些扩增子池中,我们能够从同一群体中先前测序的个体中恢复大多数单倍型。此外,我们分析了来自德国节节麦种群的一个广泛收集的新池,发现起源于独立起源的软选择的 TSR 突变很常见。向前时间模拟表明,即使在相对较低的频率和没有选择的情况下,TSR 单倍型也将持续存在数十年,这突出了准确测量 TSR 单倍型流行率对杂草管理的重要性。