ddRAD-seq of Pelteobagrus vachelli: adult male tail fin
Briefly, 500 ng of DNA template from each individual was double-digested using the restriction enzymes EcoRI and NlaIII (20 U/ reaction; New England Biolabs, Ipswich, MA, USA) in a combined reaction for 30 min at 37 C. Subsequently, each fragmented sample was purified using a Qiagen MinElute Reaction Cleanup Kit (Qiagen, Valencia, CA, USA) and eluted in 20 L of elution buffer (EB). The fragments were then ligated to P1 (includes a unique 4- to 8-bp multiplex identifier used to distinguish each individual) and P2 adapters that bound to the EcoRI and NlaIII overhangs, respectively. In each 40-L reaction, 500 ng of DNA, 1 L of P1 adapter (10 mM), 1 L of P2 adapter (10 mM), 1 L of T4 ligase (1000 U/mL), 4 L of 10X T4 ligation buffer, and double-distilled water were mixed. The ligation was performed in a PCR machine using the following conditions: 37 C for 30 min, 65 C for 10 min, followed by a decrease in temperature to 20 C at a rate of 1.3 C/min. The samples were pooled and size-selected (400-600 bp) from an agarose gel. The DNA product was subsequently purified using a Qiagen MinElute Gel Purification Kit and eluted in 10 L of EB. The paired-end (150 bp) sequencing of the ddRAD products from the 202 individuals was performed using the Illumina HiSeqXten sequencing platform (Illumina, Inc., San Diego, CA, USA). The sequencing data for each individual was extracted according to the specific multiplex identifier.