GBS libraries were prepared following Heffelfinger et al. (2014) (link). The methylation-sensitive restriction enzyme HincII [R0103; New England Biolabs (NEB)], which recognizes a degenerate six-bp sequence, was used for digestion. Libraries were constructed for the 169 F1 progenies and the two parents and were sequenced as 75-bp paired-end reads on the Illumina HiSequation 2500 in rapid run mode by the Yale Center for Genome Analysis (http://medicine.yale.edu/keck/ycga/index.aspx) following the manufacturer’s protocol. Depths of coverage for each sample are provided in Supplemental Material, Table S1. De novo SNP discovery and genotype calling was conducted using the Tassel 3.0 Universal Network Enabled Analysis Kit (UNEAK) pipeline (Lu et al. 2013 (link)). A greater number of reads are required to make accurate genotypic calls in tetrasomic polyploid populations than diploid populations. Thus, strict genotype calling thresholds were employed following the recommendations of Li et al. (2014) (link) in order to reliably distinguish between homozygotes (AAAA) and triplex heterozygotes (AAAB).