Multiplexing with three-primer PCR for rapid and economical microsatellite validation

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dc.contributor.author Vartia, Salla
dc.contributor.author Collins, Patrick C.
dc.contributor.author Cross, Thomas F.
dc.contributor.author Fitzgerald, Richard D.
dc.contributor.author Gauthier, David T.
dc.contributor.author McGinnity, Philip
dc.contributor.author Mirimin, Luca
dc.contributor.author Carlsson, Jens
dc.date.accessioned 2018-03-28T10:10:31Z
dc.date.available 2018-03-28T10:10:31Z
dc.date.issued 2014-07-07
dc.identifier.citation Vartia, S., Collins, P. C., Cross, T. F., Fitzgerald, R. D., Gauthier, D. T., McGinnity, P., Mirimin, L. and Carlsson, J. (2014) 'Multiplexing with three-primer PCR for rapid and economical microsatellite validation', Hereditas, 151(2-3), pp. 43-54. doi:10.1111/hrd2.00044 en
dc.identifier.volume 151 en
dc.identifier.issued 2-3 en
dc.identifier.startpage 43 en
dc.identifier.endpage 54 en
dc.identifier.issn 0018-0661
dc.identifier.issn 1601-5223
dc.identifier.uri http://hdl.handle.net/10468/5709
dc.identifier.doi 10.1111/hrd2.00044
dc.description.abstract 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 FST between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global FST 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. en
dc.description.sponsorship Marine Institute (Marine Research Award in Fish Population Genetics); Marine Institute and European Regional Development Fund (Grant-aid agreement no. PBA/AF/07/004, EIRCOD) en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher John Wiley & Sons Ltd. en
dc.rights © 2014, the Authors. This is an Open Access article, made available under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. en
dc.rights.uri https://creativecommons.org/licenses/by/4.0/ en
dc.subject Polymorphic microsatellite markers en
dc.subject Gadus morhua en
dc.title Multiplexing with three-primer PCR for rapid and economical microsatellite validation en
dc.type Article (peer-reviewed) en
dc.internal.authorcontactother Philip McGinnity, Biological, Earth and Environmental Sciences, University College Cork, Cork, Ireland. +353-21-490-3000 Email: p.mcginnity@ucc.ie en
dc.internal.availability Full text available en
dc.date.updated 2018-03-14T11:11:40Z
dc.description.version Published Version en
dc.internal.rssid 277098900
dc.contributor.funder Marine Institute en
dc.contributor.funder European Regional Development Fund en
dc.description.status Peer reviewed en
dc.identifier.journaltitle Hereditas en
dc.internal.copyrightchecked Yes en
dc.internal.licenseacceptance Yes en
dc.internal.IRISemailaddress P.McGinnity@ucc.ie en


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© 2014, the Authors. This is an Open Access article, made available under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Except where otherwise noted, this item's license is described as © 2014, the Authors. This is an Open Access article, made available under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.
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