GBS Library Construction and Sequencing

Preliminary testing with three restriction enzymes (PstI, MspI and ApeK1) for fragment size range led to selection of ApeKI for GBS library construction. Three GBS libraries were constructed at the USDA-ARS National Clonal Germplasm Repository (NCGR) and one was constructed at Clemson University according to the procedure previously described (Elshire et al., 2011 (link)) for 96 samples using DNA (100 ng per sample) digested with 4 U of ApeKI (New England Biolabs, Ipswich, MA, USA). The annealed and normalized unique and four-nucleotide-barcoded adaptors were obtained from Clemson University Genomics Institute (CUGI) and from the Oregon State University (OSU) Center for Genome Research and Biocomputing (CGRB) core facility. Two libraries were sequenced at the CGRB, one at CUGI, and one at the North Carolina State University Genomic Sciences Laboratory (Table S1). At each of these labs, libraries were quantitated with a Qubit^® fluorometer (Invitrogen, Carlsbad, CA, USA), checked for adequate size distribution (150–350 bp) with the Bioanalyzer 2100 HS-DNA chip (Agilent Technologies, Santa Clara, CA, USA), and sequenced with the Illumina HiSeq2000 (101 bp, single-end).