design: 2b-RAD library Construction: 1. DNA was digested with BcgI at 37 °C for 4 hours, and then heat-inactivated at 65 °C or 20 min 2. Ligation reaction with the selective adapters (5’-NNT-3’ overhangs) at 16 °C for 8 hours 3. PCR amplification was performed for the ligation fragments using a set of four primers that introduce sample-specific barcode and sequencing primers.  4. The amplification products at ~170 bp were purified via retrieval from 8% polyacrylamide gels.  5. Quantification was performed for each library and the pooled 2b-RAD library was obtained for single-end sequencing on the Illumina HiSeq2500 SE50 platform. Note: The library was constructed following the protocol published by Wang et al. (2012) with some modifications. Wang, S., Meyer, E., McKay, J. K. and Matz, M. V. 2012, 2b-RAD: a simple and flexible method for genome-wide genotyping, Nature methods, 9, 808-810. 