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建立一个可转移和具有成本效益的植物 AFLP 协议。

Towards a transferable and cost-effective plant AFLP protocol.

机构信息

Centre for Invasion Biology, Stellenbosch University, Stellenbosch, Western Cape, South Africa.

出版信息

PLoS One. 2013 Apr 16;8(4):e61704. doi: 10.1371/journal.pone.0061704. Print 2013.

Abstract

Amplified fragment length polymorphism (AFLP) is a powerful fingerprinting technique that is widely applied in ecological and population genetic studies. However, its routine use has been limited by high costs associated with the optimization of fluorescently labelled markers, especially for individual study systems. Here we develop a low-cost AFLP protocol that can be easily transferred between distantly related plant taxa. Three fluorescently labelled EcoRI-primers with anchors that target interspecifically conserved genomic regions were used in combination with a single non-labelled primer in our AFLP protocol. The protocol was used to genotype one gymnosperm, two monocot and three eudicot plant genera representing four invasive and four native angiosperm species (Pinus pinaster (Pinaceae), Pennisetum setaceum and Poa annua (Poaceae), Lantana camara (Verbenaceae), Bassia diffusa (Chenopodiaceae), Salvia lanceolata, Salvia africana-lutea, and Salvia africana-caerulea (Lamiaceae)). Highly polymorphic and reproducible genotypic fingerprints (between 37-144 polymorphic loci per species tested) were obtained for all taxa tested. Our single protocol was easily transferred between distantly related taxa. Measures of expected heterozygosity ranged from 0.139 to 0.196 for P. annua and from 0.168 to 0.272 for L. camara which compared well with previously published reports. In addition to ease of transferability of a single AFLP protocol, our protocol reduces costs associated with commercial kits by almost half. The use of highly conserved but abundant anchor sequences reduces the need for laborious screening for usable primers that result in polymorphic fingerprints, and appears to be the main reason for ease of transferability of our protocol between distantly related taxa.

摘要

扩增片段长度多态性(AFLP)是一种强大的指纹技术,广泛应用于生态和种群遗传学研究。然而,其常规使用受到与荧光标记物优化相关的高成本的限制,尤其是对于个别研究系统。在这里,我们开发了一种低成本的 AFLP 方案,可以很容易地在亲缘关系较远的植物分类群之间转移。我们的 AFLP 方案中使用了三个带有针对种间保守基因组区域的锚的荧光标记 EcoRI-引物,以及一个单非标记引物。该方案用于对四个入侵和四个本地被子植物物种的一个裸子植物、两个单子叶植物和三个双子叶植物属进行基因分型(Pinaceae 的 Pinus pinaster、Poaceae 的 Pennisetum setaceum 和 Poa annua、Verbenaceae 的 Lantana camara、Chenopodiaceae 的 Bassia diffusa、Lamiaceae 的 Salvia lanceolata、Salvia africana-lutea 和 Salvia africana-caerulea)。所有测试的分类群都获得了高度多态和可重复的基因型指纹(每个物种测试有 37-144 个多态性位点)。我们的单一方案很容易在亲缘关系较远的分类群之间转移。P. annua 的预期杂合度从 0.139 到 0.196 不等,L. camara 的预期杂合度从 0.168 到 0.272 不等,与以前发表的报告相比相当。除了单个 AFLP 方案易于转移外,我们的方案还将与商业试剂盒相关的成本降低了近一半。使用高度保守但丰富的锚序列减少了对可用引物进行繁琐筛选的需求,这些引物可产生多态性指纹,这似乎是我们的方案在亲缘关系较远的分类群之间易于转移的主要原因。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb92/3628351/92deee421163/pone.0061704.g001.jpg

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