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

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dc.contributor.authorVartia, Salla
dc.contributor.authorCollins, Patrick C.
dc.contributor.authorCross, Thomas F.
dc.contributor.authorFitzgerald, Richard D.
dc.contributor.authorGauthier, David T.
dc.contributor.authorMcGinnity, Philip
dc.contributor.authorMirimin, Luca
dc.contributor.authorCarlsson, Jens
dc.contributor.funderMarine Instituteen
dc.contributor.funderEuropean Regional Development Funden
dc.date.accessioned2018-03-28T10:10:31Z
dc.date.available2018-03-28T10:10:31Z
dc.date.issued2014-07-07
dc.date.updated2018-03-14T11:11:40Z
dc.description.abstractThe 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.sponsorshipMarine 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.description.statusPeer revieweden
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationVartia, 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/hrd2.00044en
dc.identifier.doi10.1111/hrd2.00044
dc.identifier.endpage54en
dc.identifier.issn0018-0661
dc.identifier.issn1601-5223
dc.identifier.issued2-3en
dc.identifier.journaltitleHereditasen
dc.identifier.startpage43en
dc.identifier.urihttps://hdl.handle.net/10468/5709
dc.identifier.volume151en
dc.language.isoenen
dc.publisherJohn 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.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subjectPolymorphic microsatellite markersen
dc.subjectGadus morhuaen
dc.titleMultiplexing with three-primer PCR for rapid and economical microsatellite validationen
dc.typeArticle (peer-reviewed)en
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