Genemapper 3

The possibility of formation of secondary structures among the primers was tested in AutoDimer Software [57 (link)]. A PCR reaction consisting of the simultaneous amplification of 12 markers was standardized to a final volume of 9.5 μL, using 5.0 μL 2X Qiagen Multiplex PCR Master Mix (Qiagen), 1.0 μL of Q-Solution (Qiagen), 2.0 μL of H2O, 0.5 μL of primer mix, and 1.0 μL of genomic DNA. The relative proportion of each primer in the primer mix (made up of 100 μM solutions) is listed in Table 1. The reactions were optimized to amplify 5 ng of genomic DNA.
Amplification reactions were performed in a Veriti thermocycler (Applied Biosystems). The thermocycling conditions were: initial denaturation at 95°C for 15 min, followed by 10 cycles at 94°C for 30 s, 60°C for 90 s, and 72°C for 60 s; 20 cycles at 94°C for 30 s, 58°C for 90 s, and 72°C for 60 s, and a final extension at 72°C for 60 min, 10° for min. Then, 1 μL of the amplification resulting solution was mixed with 8.5 μL of Hi-Di deionized formamide (Applied Biosystems), and 0.5 μL of GeneScan 500 LIZ (Applied Biosystems) as a molecular weight standard. The final product was analyzed using an ABI 3130 Genetic Analyzer (Applied Biosystems). The determination of fragment size and allele designation was done with the GeneMapper 3.7 software (Applied Biosystems).

We followed a standard Chelex protocol (Walsh, Metzger, & Higuchi, 1991) for extracting DNA from fry tissue and an ammonium acetate precipitation protocol (Sambrook, Fritsch, & Maniatis, 1989) for fin clips taken from adults. All adults and most fry were genotyped at 14 microsatellite loci (Table 1) though four broods (138 fry in total) were genotyped at nine microsatellite loci (multiplex 1 only). We used 2.5 or 4 μl Qiagen Type‐it Multiplex PCR Master Mix for the multiplex PCRs (total PCR volume was 6 or 8 μl respectively) and the following PCR program parameters: 5 min at 95°C, followed by 28 cycles with 30 s at 95°C, 90 s at 54°C and 30 s at 72°C, final extension at 60°C for 30 min. We scored allele sizes against an internal size standard (GeneScan‐500 ROX; Applied Biosystems) with an ABI 3130xl automatic sequencer (Applied Biosystems) and genemapper 3.7 software (Applied Biosystems, Vienna, Austria).

Twelve microsatellite loci were analyzed (Agig13519, Agig50571, Agig58115, Agig08356, Agig67103, Agig93614, Agig33291, Agig90836, Agig05001, Agig70664, Agig08912, and Agig06409), developed and selected as the most polymorphic loci for A. gigas by [13 (link)], and these loci were mixed in a multiplex-PCR system.
The multiplex PCR was run in a final volume of 9.5 µL, using a 5.0 µL 2X Qiagen Multiplex PCR Master Mix (Qiagen, Hilden, Germany), 1.0 µL of Q-Solution (Qiagen), 2.0 µL of H2O, 0.5 µL of primer mix, and 1.0 µL of genomic DNA, described by [13 (link)]. Amplification reactions were performed in a Veriti thermocycler (Applied Biosystems, Waltham, Massachusetts, United States). The thermocycling conditions were as follows: initial denaturation at 95 °C for 15 min, followed by 10 cycles at 94 °C for 30 s, 60 °C for 90 s, and 72 °C for 60 s; 20 cycles at 94 °C for 30 s, 58 °C for 90 s, and 72 °C for 60 s, and a final extension at 72 °C for 60 min, 10 °C for 5 min.
The samples were genotyped in an ABI 3130 Genetic Analyzer (Applied Biosystems Inc., Foster, CA, USA) using a mixture of 1 μL of the PCR product, 8.5 μL of formaldehyde and 0.5 μL of GeneScan 500 LIZ (Applied Biosystems). The determination of fragment size and allele designation was conducted with the GeneMapper 3.7 software (Applied Biosystems).