Vartia Salla, Collins Patrick C, Cross Thomas F, Fitzgerald Richard D, Gauthier David T, McGinnity Philip, Mirimin Luca, Carlsson Jens
Carna Research Station, Ryan Institute, National University of Ireland, Galway, Carna, Connemara, Co. Galway, Ireland; Area 52 Research Group, School of Biology & Environment Science, University College Dublin, Belfield, Dublin, Ireland.
Hereditas. 2014 Jun;151(2-3):43-54. doi: 10.1111/hrd2.00044.
The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus F(ST) between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global F(ST) was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.
新一代测序技术的革命使得基因标记能够被快速发现，然而，开发功能完备的新标记仍然需要漫长且成本高昂的标记验证过程。本研究报告了一种快速且经济的方法，用于验证和应用从大西洋鳕鱼（Gadus morhua，Linnaeus 1758）的454焦磷酸测序文库中获得的多态微卫星标记。从原始读数设计引物，以扩增特定的扩增子大小范围，从而实现有效的PCR多重化。多重化与一种三引物PCR方法相结合，使用四个通用尾端用不同的荧光染料标记扩增子。总共测试了192对引物，得到73个多态性标记。其中，55个位点被组合成六个多重面板，每个面板包含6至11个标记。在先前研究中显示出遗传分化的凯尔特海（n = 46）和斯科舍陆架（n = 46）的大西洋鳕鱼群体中评估了这些位点的变异性。两个样本之间的多位点F(ST)估计为0.067（P = 0.001）。在排除三个可能处于选择状态的位点后，全局F(ST)估计为0.043（P = 0.001）。我们的技术结合了三引物和多重PCR技术，允许同时筛选和验证相对大量的微卫星位点。
