PDF(Pubmed)
Abstract：
Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.
摘要：
微卫星基因座的多重聚合酶链反应（PCR）允许在单个PCR反应中同时扩增两对或更多对引物；因此，它是成本和时间有效的。然而,在非模型物种中很少有尝试的报道。在这项研究中,通过结合基于基因组的从头开发和跨物种应用方法，一个多重PCR系统，包括由26个新基因组和7个文献来源的基因座组成的33个微卫星的5个PCR反应，进行了多态性测试，跨物种可转移性，以及评估三种核桃物种的遗传多样性和种群结构的能力（Juglansspp。).我们发现基于基因组的方法比其他方法更有效。为每个基因座开发了等位基因梯以增强实验室之间一致的基因分型。种群遗传分析结果表明,33个基因座均成功跨3个物种转移,表现出较高的多态性和较强的遗传结构。因此，多重PCR系统在核桃品种中具有很高的适用性。此外，我们提出了一个有效的管道来表征和基因型多态微卫星基因座。这里开发的新工具箱将有助于核桃未来的生态学和进化研究，并可以作为其他植物物种的模型。
