*4.9. DNA Preparation, PCR Amplification, and Agarose Gel Electrophoresis for CAPS Markers*

Either buds, leaves, or cambium were prepared for extraction of total DNA following a slightly modified ATMAB protocol according to Dumolin et al. [82]. The type of tissue was dependent on what was provided as reference material used in this study.

PCR amplification was performed in 20 μL volume with 20 ng DNA. For all markers shown in Table 1, the reaction mixture contains 1× BD Buffer, 2 mM MgCl2, 200 μM each dNTP, 1xDMSO (NEB), 0.2 μM of each Primer, and 1 unit Taq Polymerase (DCS Pol, DNA Cloning Service, Hamburg), except for marker 5\_Fagales\_*mat*R (1.5 unit Taq polymerase was used). The PCR was performed with the following program: 94 ◦C for 4 min, followed by 35 cycles with 94 ◦C for 45 s, 58 ◦C for 1 min, 72 ◦C for 1 min, and additional 5 min at 72 ◦C final elongation at the end.

For the restriction analyses for each marker, 10 μL PCR product was used in a volume of 20 μL. The restriction analyses (in deviation from the manufacturer's protocols) were performed as follows: the PCR product amplified with the marker 5\_Fagales\_*matR* was digested with 4 units of the enzyme *Nci*I for 8 h at 37 ◦C and 20 min of inactivation at 80 ◦C—the same conditions were used for 3\_Fagus\_*matR* using the enzyme *Bst*XI; 4\_Fagaceae\_*nad7* was digested with the enzyme *Sfc*I with the same conditions as the former; 3\_Fagus\_*ccmFc* was digested with 5 units of the enzyme *Bsm*BI for 8 h at 55 ◦C and 20 min of inactivation at 80 ◦C. Restriction products were visualized relative to a 50 bp ladder (Life technologies, Germany, Martinsried) using a 1.5% agarose gel stained with Roti-Safe Gelstain (Carl Roth, Germany, Karlsruhe).
