Gs500liz size standard

DNA was extracted from blood and tissue samples either with the standard phenol–chloroform method [50 ], or using the UltraClean® BloodSpin™ Kit or UltraClean® Tissue & Cells DNA Isolation Kit (MoBio Laboratories), and from feathers using the method described in Rönkä et al. [34 (link)]. Individuals were genotyped for 12 microsatellite loci, which were amplified in 10 µl volumes containing 20–100 ng of template DNA, 0.1 µM of each primer, 0.8–1 mM MgCl₂, 0.2 mM of dNTPs, 1 µl of 10 × PCR-Buffer and 0.l U of DNA-polymerase (Biotools). The amplification profile was 94 °C for 1 min followed by 35 cycles of 94 °C for 30 s, 52–58 °C for 45 s (see Additional file 1: Table S1), 72 °C for 45 s and 72 °C for 10 min for final extension. The PCR reactions were run with ABI 3730 sequencer using GS500-Liz size standard (Applied Biosystems) and the loci were scored with GeneMapper v. 4.0. (Applied Biosystems), except for the Swedish samples, which were scored with CEQ^TM8000 Genetic Analysis System (Beckman Coulter) using the Fragment Analysis Module v. 8.0.52. Due to possible differences between the allele sizes defined by the two sequencers, samples were calibrated by genotyping five Swedish individuals with both sequencers. Genotyping error rate was calculated by amplifying most individuals twice. If differences were found between the two runs, samples were genotyped twice more.

For each of the 14 SSR markers, the forward primer was labeled with either FAM, HEX, or TAMRA fluorophore. The PCR reaction system was the same as described above. PCR cycling was performed as follows: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 53, 55, or 56 °C (depending on the primer pair) for 30 s, 72 °C for 30 s, with a final extension of 10 min at 72 °C. The amplicons were separated using capillary electrophoresis. Capillary electrophoresis was performed in multiplex reactions of three amplicons with three different fluorescent dyes per reaction. The PCR products were analyzed using an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with a GS500LIZ Size Standard (Applied Biosystems). The output files were analyzed using GeneMapper v4.1 software (Applied Biosystems) to identify the lengths of amplified fragments at each locus.