Sample preparation and DNA extraction

The mixed reference population of 500 individuals was bred by a random fertilization of 30 males and 30 females at the large yellow croaker breeding base of Jimei University in Ningde, Fujian, China. All fish individuals were 1.5 year old with the total length and weight of 24.5–25.9 cm and 217.8–234.1 g (95% confidence interval), respectively. To extract genomic DNA respectively from 500 individuals, the dorsal fins (20–30 mg) of the fish individuals were collected, frozen in liquid nitrogen for the following DNA extraction. Total genomic DNA was prepared in 1.5 ml microcentrifuge tubes containing 550 µl TE buffer (100 mM NaCl, 10 mM Tris, pH 8, 25 mM EDTA, 0.5% SDS and proteinase K, 0.1 mg/ml). The samples were incubated at 55 °C overnight and subsequently extracted twice using phenol and then phenol/chloroform (1:1) method. DNA was precipitated by adding two and a half volumes of ethanol, collected by brief centrifugation, washed twice with 70% ethanol, air dried, re-dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5). DNA concentration and quality were estimated with an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and by electrophoresis in 0.8% agarose gels with a lambda DNA standard.