Genotyping by Sequencing Protocol

A tissue plug of approximately 3 mm diameter from each fin clip was used for DNA extraction. Prior to extraction, the tissue was air dried overnight to remove all traces of ethanol and then DNA was extracted following a salt-based extraction protocol (Clarke et al. 2014 (link)). A subset of the DNA extracted samples was visually assessed via 1.0% agarose gel in order to ensure the existence of high molecular weight DNA. Quantification was performed using Picogreen (Quant-iT Picogreen dsDNA Reagent, Cat P11495, Life Technologies, Carlsbad, California, United States) fluorescence. Template DNA was digested with PstI (recognizing the CTGCA|G motif) and MspI (recognizing the C|CGG motif) restriction enzymes. Subsequent library preparation followed the method outlined in Elshire et al. (2011) (link). Constructed libraries were size selected between 193 – 318 bp using a BluePippin (Sage Science). In total 10 GBS libraries were constructed containing 90 – 94 individuals each. Sequencing was performed in 5 lanes of an Illumina HiSeq 2500 using 100 cycles single end (SE) V4 chemistry.