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.