PCR and Illumina high-throughput sequencing
Based on about 200 DNA sequences from a library of expressed sequence tags of A. marina (J. Huang et al., 2014), we developed a new set of 94 pairs of primers anchored at exons but spanning at least one intron. All primers produced amplicons with length 500 to 1500 bps. All 94 amplicons from one A. marina individual were sequenced using the Sanger method to obtain reference sequences. We also did this for one A. alba individual for use as an outgroup. For the DNA mixture of each population, polymerase chain reaction (PCR) amplification was performed using each of the 94 primer pairs. To reduce amplification errors, TaKaRa high-fidelity PrimerStar HS DNA polymerase was used. The 30 μL PCR mixture consists of 3 μL 10x TaqBuffer (Mg2+), 3 μL dNTPs (2mM/μL), 1.5 μL of each primer (10μM/μL), 0.5 μL HS DNA Polymerase, 3 μL DNA template (~10ng/μL) and 19 μL deionized water. The PCR program was: 4 min at 94°C; 30 cycles of 10 s at 94°C, 30 s of annealing at the corresponding temperature (Supplementary Table 1), extension at 72°C for 2 min; followed by 8 min final extension at 72°C. Reactions were held at 16°C before PCR products were subjected to electrophoresis on 1.2% agarose gels. Target bands were excised under ultraviolet light and extracted using the Pearl DNA Gel Extraction Kit (Pearl, Guangzhou, China). Extracted DNA was examined by NanoDrop 2000 to ensure that the amount of each gene product was no less than 100ng. PCR products of the 94 loci from the same population were again pooled, using 100 ng of DNA per locus. We thus obtained 16 PCR product mixtures, each including amplicons from 94 loci.
PCR product mixtures from each population were delivered for sequencing on the Illumina HiSeq 2000 platform at BGI (Shenzhen) following the manufacturer’s instructions. DNA libraries of 200 bp were constructed for these mixtures and an 8 bp index in the adapter was used to distinguish the populations. Method details used for library construction were the same as those detailed in the Supplementary materials of our previous publication (Zixiao Guo et al., 2016). Raw reads produced were 90 or 130 bps in length.