Materials and Methods
Sampling
We collected 49 individuals of S. japonica from five populations distributed throughout the coastal waters of China and Japan (Figure 1 and Table 1). All individuals were identified on the basis of morphological features. A piece of muscle tissue was obtained from each individual and preserved in 95% of ethanol or frozen for DNA extraction.
Sequencing, read mapping, and SNP calling
Genome sequencing was performed using the Illumina HiSeq 2500 platform by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Quality control and removal of potential adaptor sequences from raw reads were performed to ensure the accuracy of bioinformatics analysis. Paired-end reads from each individual were aligned to the reference genome (unpublished data) using the Burrows–Wheeler Aligner (BWA) with the option “mem-t 4-k 32 -M” (Li & Durbin, 2009). After mapping, the resulting bam files were sorted using SAMtools (Li, Handsaker, Wysoker, Fennell, & Ruan, 2009), and duplicated reads were removed.
Raw SNPs were identified on the basis of mpileup files generated by SAMtools. The filtering threshold for raw SNPs was set to require a quality score of ≥20. Furthermore, an SNP was discarded if the total coverage of the SNP was less than one third or greater than five times the overall coverage. If two SNPs were <5 bp apart, both SNPs were removed.