DNA extraction and GBS library preparation

Healthy leaves were collected from the field-grown parents and F₂ progeny, flash-frozen in liquid nitrogen and stored at − 80 °C until further use. The leaves were ground to a fine powder using a TissueLyserII bead mill (Qiagen) and genomic DNA was isolated using a CTAB procedure. DNA quantity and quality were determined by Nanodrop spectrophotometer and agarose gel electrophoresis, respectively. DNA samples were diluted in 1/10th TE buffer to a concentration of 50 ng/µL.

DNA was digested with the restriction enzymes PstI, MspI and ApeKI, and barcoded libraries were constructed for each sample as described in Qi et al. (2018). Please note that we no longer recommend the use of a third restriction enzyme to reduce the fragment pool (Qi et al. 2018). Each library was quantified on a Qubit 2.0 fluorometer using the dsDNA High Sensitivity Assay Kit. The quality of each library was verified by agarose gel electrophoresis. Equal amounts (30 ng/sample) of 200 quality-verified samples with a concentration of at least 5 ng/µL were pooled. DNA fragments smaller than 300 bp, including primer dimers, were removed from the pooled libraries using Sera-Mag SpeedBeads (Qi et al. 2018). The pooled libraries were sequenced on an Illumina NextSeq platform (paired-end 150 bp) to yield approximately 400 Mb of sequence per sample.