DNA from young (in the end of bud burst) leaves of the ‘Improved French’ was extracted using modified protocol by Kubisiak et al.24. Briefly, nuclei were isolated using extraction buffer (0.35 M sorbitol, 10% polyethylene glycol 8000, 0.2% bovine serum albumin, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1 mM spermidine, 1 mM spermine, and 1% β-mercaptoethanol). Pelleted nuclei were washed with organelle wash buffer containing 0.35 M sorbitol, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1 mM spermidine, 1 mM spermine, and 1% β-mercaptoethanol. The DNA from nuclei was extracted by treatment with proteinase K (5 µl per 400 ul) in lysis buffer containing 0.5% N-lauryl sarcosine, 1% CTAB, 0.7 M NaCl at 65 °C for 12 min, phase-separated with equal volume of chloroform: isoamyl alcohol (24:1 vol/vol) extraction and precipitated with 2-isopropanol (1:1 vol/vol). The DNA was washed twice in 70% ethanol, dried at room temperature and resuspended in 0.01 M tris HCl, pH 8.0. Then, the DNA was treated with 3 µl of the Ambion® RNase Cocktail™ (Thermo Fisher Scientific Inc., USA), for 30 min at 37 °C, followed by chloroform: isoamyl alcohol (24:1 vol/vol) extraction and precipitation with two volumes of ethanol. Finally, the DNA was resuspended in 100 µl of 0.01 M Tris-HCl, pH 8.0. The quality and integrity of the DNA were evaluated using Qubit 2 Fluorometer (Thermo Fisher Scientific Inc., USA), a NanoDrop ND-8000 (Thermo Fisher Scientific Inc., USA) followed by electrophoresis on 1% agarose gels.

Sequencing and assembly were performed by NRGene (San Diego, CA) using a combination of Illumina™ technologies including paired-end reads, mate-pair reads of differing sizes, and sequencing and assembly of Chromium 10x libraries (Table S8). The sequencing data were processed and assembled using DeNovoMAGIC™ assembler application version 3.0. The integrity of the assembly was verified using several quality-assurance procedures including the independent BUSCO benchmark (http://busco.ezlab.org/)7 which is used to specifically indicate the genic region integrity, ploidy, and zygosity characteristics of the assembled genome. The assembled scaffolds are available at GDR (www.rosaceae.org).

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