Genotyping-by-sequencing

Young leaf tissue was harvested from each genet prior to planting, freeze dried, and genomic DNA was extracted using the BioSprint 96 Plant DNA Kit (QIAGEN, the Netherlands). DNA was quantified using QuantiFluor dsDNA System (Promega Corporation, WI, USA) and normalized to 10 ng/µl. Genotyping by sequencing libraries were developed using PstI/Msp I enzymes following Zhang et al. (2016) with two barcodes per sample. Every 96 samples were pooled, creating a total of fifteen 96-plex libraries. The common parent was sampled eight times and the donor parents six each to achieve higher sequencing depth. Libraries were amplified and cleaned using the QIAquick PCR Purification Kit (QIAGEN, the Netherlands), quality control was done using Picogreen (ThermoFisher Scientific, MA, USA), Agilent Bioanalyzer (Agilent, CA, USA) and Kapa qPCR, and subjected to size selection of 160–240bp using PippinHT (3% agarose). Each pool was sequenced in a single lane of a 100 bp single read run on the Illumina HiSeq 2500 HO using v4 chemistry at the University of Minnesota Genomics Center.