*4.2. DNA Sequencing of F. sylvatica L.*

Dormant buds of the *F. sylvatica* individual FASYL\_29 were sampled, green tissues were dissected and DNA was extracted for short read sequencing following a slightly modified ATMAB protocol based on the protocol of Dumolin et al. [82]. Standard genomic library preparation and 300 bp paired-end sequencing was performed on Illumina MiSeq at 24× coverage (GATC Biotech AG, Konstanz, Germany).

For long read sequencing, DNA was extracted using a combination of a lysis buffer, Sera-Mag Speed beads, and a purification step, adapted from [83]. Briefly, the outer layer of leaf buds was removed; then, they were cut into pieces (to facilitate grinding), collected into 2 mL Eppendorf tubes, and frozen in liquid nitrogen (10 buds in total; two buds per tube). Samples were ground using a Retsch Mill (Retsch MM300, Haan, Germany), with two stainless steel beads (5 mm) per tube, at a speed of 25 Hz for 35 s. DNA lysis was performed by adding 700 μL lysis buffer (1% PVP 40 (*w*/*v*), 1% PVP 10 (*w*/*v*), 500 mM NaCl, 100 mM Tris-HCl pH 8.0, 50 mM EDTA, 5 mM DTT, 1.25% SDS (*w*/*v*), and 1% Sodium metabisulfite (*w*/*v*)) to each of the samples, which were then mixed gently by flicking and incubated at 64 ◦C for 30 min. Following that, 1 μL RNase A (10 mg/mL) per 1 mL lysis buffer was added, and the samples were incubated at 37 ◦C for 50 min at 400 rpm on a thermomixer. After the first 20 min, 10 μL Proteinase K (800 units/mL) was added to each sample. Once the incubation time ended, samples were left on ice for 2 min to cool down, and 0.3 volume 5 M potassium acetate pH 7.5 was added. Samples were manually mixed by inversion 20 times, slowly, then centrifuged at 8000× *g* for 12 min at 4 ◦C.

For DNA size selection, the supernatant was transferred to clean 1.5 mL LoBind Eppendorf tubes and 0.8 V of a homogenized Sera-Mag Speed beads solution (10 mM Tris-HCl, 1 mM EDTA pH 8.0, 1.6 M NaCl, 11% PEG 8000, 0.4% beads (*v*/*v*)) was added, and the tubes were mixed gently by flicking. The samples were placed on a rotor for 10 min, briefly centrifuged, and placed on a magnet. Once the beads were on the back of the tubes and the solution became clear, the supernatant was discarded, and the beads were washed twice with 1 mL freshly prepared 70% ethanol. After the last ethanol wash, the tubes were removed from the magnet and briefly centrifuged. After placing them back on the magnet, the last drops of ethanol were pipetted off. The beads were air dried for 30 s; then, the tube was removed from the magnet, and 50 μL pre-heated (50 ◦C) 10 mM Tris-HCl pH 8.0 was added for elution. The tubes were flicked to resuspend the beads then incubated for 10 min at room temperature. Finally, the tubes were placed back on the magnet, and once the solution was clear, it was transferred to fresh tubes.

The DNA purification step was performed using chloroform:isoamylalcohol (24:1). Since the extraction was performed using multiple tubes, the eluted DNA from each tube (~80 μL) was pipetted into a single tube, comprising a total of 400 μL. Then, one volume of chloroform:isoamylalcohol was added, and it was mixed by inversion for 5 min on a rotor. After that, the tube was centrifuged at 5000× *g* for 2 min at room temperature, and the upper phase was transferred to a fresh tube. The chloroform:isoamylalcohol step was repeated, and after that, a 0.1 volume of 100% cold ethanol was added, and the sample was centrifuged at 5000× *g* for 2 min at room temperature. The pellet was washed with 70% ethanol and resuspended in 50 μL 10 mM Tris-HCl pH 8.0 for 2 h at room temperature. The sample was stored at 4 ◦C until library preparation.

The sequencing library was prepared using the Ligation Sequencing Kit (SQK-LSK109) following the manufacturer's instructions (Oxford Nanopore Technologies, Oxford, UK), with 2 μg DNA as input. The R9.4.1 MinION Flow Cell was primed with the Flow Cell Priming Kit (EXP-FLP002). Sequencing was performed on a MinION Mk1B device (MIN-101B) connected to a MinIT computer (MNT-001). We used the MinIT software version 19.01.1, and further basecalling was performed using guppy v3.2.2.
