PCR and Illumina high-throughput sequencing
Based on about 200 DNA sequences from a library of A. marinaexpressed sequence tags (Huang et al., 2014), we developed a new set of
primers anchored at exons but spanning at least one intron. The 94 pairs
of primers producing amplicons 500 to 1500 bps long were used in this
study. We performed polymerase chain reaction (PCR) amplification on DNA
pool from each population using our 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 (Table S1 in
the online supplementary file), 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 pools, each including amplicons from 94 loci.
PCR product pools from each population were delivered for sequencing on
the Illumina Genome Analyzer and Illumina HiSeq 2000 platform at BGI
(Shenzhen) following the manufacturer’s instructions. 200 bp DNA
libraries 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 (Guo et al., 2016).
Raw reads produced from the Illumina Genome Analyzer platform were 90
bps in length (all populations except MC, BB, and DW; abbreviations of
population names are defined in Table 1), while those from the Illumina
HiSeq 2000 platform were 130 bps in length (MC, BB, and DW).