RAD-Seq of Charybdis feriata: adult males and females muscles
each sample was digested with EcoRI (recognition site 5  -G/AATTC- 3 ) high fidelity restriction enzyme. Individual specific P1 adapters, each with a unique barcode, were ligated to the EcoRI digested DNA with T4 DNA Ligase. Samples were pooled by sex into separate male and female libraries and randomly sheared by Bioruptor to an average size of 350 bp, and the sheared DNA was then purified with a QIAquick PCR Purification Kit (Qiagen). Samples were size separated (300-500bp) using gel electrophoresis, and the appropriate fragments were purified using a MiniElute Gel Extraction Kit (Qiagen). Then the end repair, dA overhang addition, purification and P2 paired-end adapter ligation were conducted as the original RAD protocol. After purification, the products were PCR amplified via 13-15 cycles. The PCR products were size selected (300-500 bp) by gel electrophoresis, and purified as described as above. The purified products were quality assessed by electrophoresis and accurately quantified by fluorimetry (Qubit). Each library was pooled with an equal amount to make a final library that was sequenced (150 base pair paired-end reads) in a lane of the Illumina HiSeq 3000 platform following standard protocols at Shanghai genenergy Biotechnology Co., Ltd (Shanghai, China).