Multiplexing with three-primer pcr for rapid and economical microsatellite validation
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 polym...
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ftnuigalway:oai:https://researchrepository.universityofgalway.ie:10379/14278 2024-09-30T14:32:09+00:00 Multiplexing with three-primer pcr for rapid and economical microsatellite validation Vartia, Salla Collins, Patrick C. Cross, Thomas F. Fitzgerald, Richard D. Gauthier, David T. McGinnity, Philip Mirimin, Luca Carlsson, Jens 2014-06-01 http://hdl.handle.net/10379/14278 https://doi.org/10.13025/25953 https://doi.org/10.1111/hrd2.00044 unknown Springer Nature Hereditas Vartia, Salla; Collins, Patrick C. Cross, Thomas F.; Fitzgerald, Richard D.; Gauthier, David T.; McGinnity, Philip; Mirimin, Luca; Carlsson, Jens (2014). Multiplexing with three-primer pcr for rapid and economical microsatellite validation. Hereditas 151 (2), 43-54 0018-0661 http://hdl.handle.net/10379/14278 https://doi.org/10.13025/25953 doi:10.1111/hrd2.00044 Attribution-NonCommercial-NoDerivs 3.0 Ireland https://creativecommons.org/licenses/by-nc-nd/3.0/ie/ cod gadus-morhua simple sequence repeats atlantic cod population-structure tailed primers nonmodel organisms marker development genetic-analysis genome size loci Article 2014 ftnuigalway https://doi.org/10.13025/2595310.1111/hrd2.00044 2024-09-17T14:44:29Z 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. Article in Journal/Newspaper atlantic cod Gadus morhua National University of Ireland (NUI), Galway: ARAN |
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Open Polar |
collection |
National University of Ireland (NUI), Galway: ARAN |
op_collection_id |
ftnuigalway |
language |
unknown |
topic |
cod gadus-morhua simple sequence repeats atlantic cod population-structure tailed primers nonmodel organisms marker development genetic-analysis genome size loci |
spellingShingle |
cod gadus-morhua simple sequence repeats atlantic cod population-structure tailed primers nonmodel organisms marker development genetic-analysis genome size loci Vartia, Salla Collins, Patrick C. Cross, Thomas F. Fitzgerald, Richard D. Gauthier, David T. McGinnity, Philip Mirimin, Luca Carlsson, Jens Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
topic_facet |
cod gadus-morhua simple sequence repeats atlantic cod population-structure tailed primers nonmodel organisms marker development genetic-analysis genome size loci |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Vartia, Salla Collins, Patrick C. Cross, Thomas F. Fitzgerald, Richard D. Gauthier, David T. McGinnity, Philip Mirimin, Luca Carlsson, Jens |
author_facet |
Vartia, Salla Collins, Patrick C. Cross, Thomas F. Fitzgerald, Richard D. Gauthier, David T. McGinnity, Philip Mirimin, Luca Carlsson, Jens |
author_sort |
Vartia, Salla |
title |
Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
title_short |
Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
title_full |
Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
title_fullStr |
Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
title_full_unstemmed |
Multiplexing with three-primer pcr for rapid and economical microsatellite validation |
title_sort |
multiplexing with three-primer pcr for rapid and economical microsatellite validation |
publisher |
Springer Nature |
publishDate |
2014 |
url |
http://hdl.handle.net/10379/14278 https://doi.org/10.13025/25953 https://doi.org/10.1111/hrd2.00044 |
genre |
atlantic cod Gadus morhua |
genre_facet |
atlantic cod Gadus morhua |
op_relation |
Hereditas Vartia, Salla; Collins, Patrick C. Cross, Thomas F.; Fitzgerald, Richard D.; Gauthier, David T.; McGinnity, Philip; Mirimin, Luca; Carlsson, Jens (2014). Multiplexing with three-primer pcr for rapid and economical microsatellite validation. Hereditas 151 (2), 43-54 0018-0661 http://hdl.handle.net/10379/14278 https://doi.org/10.13025/25953 doi:10.1111/hrd2.00044 |
op_rights |
Attribution-NonCommercial-NoDerivs 3.0 Ireland https://creativecommons.org/licenses/by-nc-nd/3.0/ie/ |
op_doi |
https://doi.org/10.13025/2595310.1111/hrd2.00044 |
_version_ |
1811636395102437376 |