2.2 | RAD library construction and sequencing
The total DNA of each individual was extracted from the muscle tissue
using the standard phenolchloroform method (Sambrook et al., 1989). DNA
quality was first assessed by visually inspecting for signs of
fragmentation on 1% agarose gels. To avoid the potential
disproportionate representation of individuals in sequencing pools owing
to the varying DNA quality across samples, we reextracted and reexamined
any DNA samples showing degradation on agarose gels. Only nondegraded
samples were processed further and quantified (twice) with a NanoDrop
ND-1000 spectrophotometer. Each sample was then diluted to 10 ng/μL,
requantified and pooled for each population. Next, each pooled sample
was quantified (twice) with the spectrophotometer and equalized to 10
ng/μL to construct the RAD library.
To construct RAD libraries, DNA was fragmented by the restriction enzymeEco RI. A P1 adapter including a forward amplification primer,
sequencing primer, and an 8-base pair (bp)-specific barcode was added to
each of the fragmented DNA pools. The barcoded samples were then pooled
and sheared randomly, and a P2 adapter was added to the sheared DNA
fragments. DNA with a P1 adapter was selectively enriched by PCR
amplification. Finally, DNA fragments of 300–500 bp were gel-purified
and sequenced on the Illumina HiSeq2000 platform to generate 100-bp
paired-end reads. All pools were sequenced on a paired-ends sequencing
lane. The construction of RAD libraries and sequencing were carried out
by BGI Tech Solutions Co., Ltd. (Shenzhen, China). Filtered reads for
the RAD data pertaining to this study can be accessed through the NCBI
GenBank Short Read Archive (PRJNA990519).