Open AccessMultiplexing with three‐primer PCR for rapid and economical microsatellite validation
Author(s) -
Vartia Salla,
Collins Patrick C.,
Cross Thomas F.,
Fitzgerald Richard D.,
Gauthier David T.,
McGinnity Philip,
Mirimin Luca,
Carlsson Jens
Publication year - 2014
Publication title -
hereditas
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 50
eISSN - 1601-5223
pISSN - 0018-0661
DOI - 10.1111/hrd2.00044
Subject(s) - biology , microsatellite , amplicon , primer (cosmetics) , multiplex , multiplex polymerase chain reaction , genetics , pyrosequencing , genotyping , gadus , genetic marker , polymerase chain reaction , computational biology , genotype , gene , fishery , allele , chemistry , organic chemistry , fish <actinopterygii>
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.