Genotyping

Leaf tissue was collected using a hole punch from each accession in each cross once, and collected in duplicate from both sets of parents. Punched tissue was dried using a freeze dryer before being shipped to the AAFC Kentville Research and Development Centre in Kentville, Nova Scotia, Canada for DNA extraction. Tissue was lyophilized and then ground using a 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA). Whole-genome DNA was extracted using a NucleoSpin 96 Plant II kit (Machery-Nagel, Düren, Germany) with the following modifications to the kit protocol: samples were incubated in lysis buffer for 60 min, and were processed using a vacuum manifold with an additional step of filtering lysate through receiver plates before proceeding to the binding step. DNA samples were quantified using the QuantiFluor dsDNA System and the GloMax-Multi+Microplate Multimode Reader with Instinct (Promega, Madison, WI, USA).

Library preparation and DNA sequencing were performed at L’Institut de Biologie Intégrative et des Systèmes (IBIS) at Université Laval, Quebec City, Québec, Canada using an Illumina HiSeq 2000 and the GBS approach,²⁶ with the restriction enzyme ApeKI.³⁴ Each F₁ progeny was sequenced once, while each parent in each cross was sequenced in duplicate as recommended by Gardner et al.³⁵