title: Multiplexing with three-primer PCR for rapid and economical microsatellite validation.
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 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.
authors.affiliation: 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. salla.vartia@gmail.com.
authors.forename: Salla
authors.initials: S
authors.lastname: Vartia
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Collins
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Cross
authors.forename: Richard D
authors.initials: RD
authors.lastname: Fitzgerald
authors.forename: David T
authors.initials: DT
authors.lastname: Gauthier
authors.forename: Philip
authors.initials: P
authors.lastname: McGinnity
authors.forename: Luca
authors.initials: L
authors.lastname: Mirimin
authors.forename: Jens
authors.initials: J
authors.lastname: Carlsson
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D048389:Gadus morhua / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics*; D018895:Microsatellite Repeats / Q000235:genetics*; D009792:Oceans and Seas; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D054928:Validation Studies as Topic
chemical_list: D017931:DNA Primers