Microsatellite Markers for Salmonid Genome Analysis

A total of 1435 microsatellite markers were developed or obtained from the literature including anonymous markers [19 (link),59 (link),61 (link),67 (link)-80 (link)], markers developed in other salmonids [62 (link),81 (link)-83 (link)], markers identified from BACs either containing genes or cytogenetic assignments [60 (link),84 (link),94 (link)-96 (link)], or markers representing expressed sequence tags (ESTs) and serving as comparative loci with sequenced genomes of model fish species [80 (link),85 (link)]. Marker information including locus names, optimum annealing temperatures and magnesium concentrations, GenBank accession numbers, and primer sequences are reported in Additional File 1, Worksheet 1. Markers were either genotyped using the tailed protocol of Boutin-Ganache et al. [97 (link)] or by direct fluorescent labelling (with FAM, HEX, or NED) of the forward primer according to manufacturer protocols (ABI, Foster City, CA, USA). Primer pairs were obtained from commercial sources (forward primers labelled with FAM or HEX from Alpha DNA, Montreal, Quebec, Canada, or NED from ABI, Foster City, CA, USA). PCR reactions consisted of 12 μl reaction volumes containing 12.5 ng DNA, 1.5–2.5 mM MgCl₂, 1.0 μM of each primer, 200 μM of dNTPs, 1× manufacturer's reaction buffer and 0.5 units Taq DNA polymerase. Thermal cycling consisted of an initial denaturation at 95°C for 15 min followed by 30 cycles of 95°C for 1 min, annealing temperature for 45 s, 72°C extension for 45 s, then a final extension at 72°C for 10 min. PCR products were visualized on agarose gels after staining with ethidium bromide. Markers were grouped in combinations of two or three markers based on differences in fluorescent dye color and amplicon size. Three μl of each PCR product was added to 20 μl of water, 1 μl of the diluted sample was added to 12.5 μl of loading mixture made up with 12 μl of HiDi formamide and 0.5 of Genscan 400 ROX internal size standard. Samples were denatured at 95°C for 5 min and kept on ice until loading on an automated DNA sequencer ABI 3730 DNA Analyzer (ABI, Foster City, CA, USA). Output files were analyzed using GeneMapper version 3.7 (ABI, Foster City, CA, USA), formatted using Microsoft Excel and stored in Microsoft Access. As a result of the evolutionarily recent genome duplication, microsatellite markers in salmonids are often present in two copies in the genome, each copy potentially having overlapping allele size ranges and possibly including alleles having identical sizes. Markers which were duplicated were scored as independent loci, adding an "a" and "b" to differentiate their locus names. Duplicated loci with overlapping and/or identical allele sizes were scored only in the family containing the most informative meiosis.