2.3. DNA extraction, mitochondrial DNA sequencing, microsatellite development, and genotyping

Total genomic DNA was extracted from a piece of foot muscle using a standard phenol chloroform method. The amplification of the complete mitochondrial NADH dehydrogenase subunit 6 (mt ND6) gene was performed by PCR in a 25‐μl reaction mixture containing template DNA (1 μl), ddH2O (10.5 μl), 2X PCR Mix (12.5 μl), and a pair of “Littorina specific” primers [Lbnd‐F (5’‐AGG TAC ATA TTC CTG CGC TCT GAA A‐3’) and Lbnd‐R (5’‐GTG TGC GCA TGA AAT GTA T‐3’)] (10 μM, respectively) (Kim et al., 2003). The thermal cycling profile included precycling denaturation at 95 ℃ for 5 min, followed by 35 cycles of denaturation at 95 ℃ for 30 s, annealing at 50 ℃ for 40 s, extension at 72 ℃ for 1 min, and a final extension at 72 ℃ for 10 min. The PCR products were checked by electrophoresis on 2% agarose. Then, PCR products were gel purified using gel extraction kit and cycle‐sequenced using the BigDye™ Terminator Cycle Sequencing Kit V. 3.1 on an ABI 3730xl DNA Analyzer (Applied Biosystems) at biotechnology company TingKe (Qingdao, China). All the samples were sequenced with both forward and reverse primers, which were the same as those for PCR amplification.

For the development of microsatellite loci, a de novo genomic library with insert size ~350bp was constructed following a standard Illumina protocol. Sequencing was performed to generate 150‐bp paired‐end reads on the HiSeq X Ten platform (Illumina) at Novogene Bioinformatics Technology (Tianjin, China). Then, de novo assembly was carried out using MEGAHIT using default settings (Li, Liu, et al., 2015). The scaffolds were then used for further microsatellite isolation and discovery. Microsatellite discovery and primer design were performed with QDD 3.1.2 (Meglécz et al., 2010) on a local Galaxy platform. Sequences with microsatellites (pure tandem repeats of di‐ to hexa‐nucleotide motif with at least ten uninterrupted repeats) and with at least 150 bases of flanking sequences on both sides were detected for primers design in Primer3 (Rozen & Skaletsky, 2000). Finally, a total of 12 newly developed polymorphic microsatellite loci (Table S1) were used for subsequent analysis. The amplification of the 12 microsatellite loci was performed by the PCR protocol described in Liu and Avise (2011). The thermal cycling parameters were the same as that previously described for mt ND6 except annealing temperatures (53–58℃ for different loci (Table S1)). PCR products with different fluorescent labels were pooled and electrophoresed on an ABI 3730xl automated sequencer (Applied Biosystems) at Sangon Biological Technology (Qingdao, China). Allele sizes were determined with the GS500‐ROX size standard using GeneMarker 2.2.0 (SoftGenetics, State College, USA).