2.5. DNA extraction and genotyping

Total DNA was extracted using a modified cetyl‐trimethylammonium bromide (CTAB) protocol, described previously by Drori et al. (2017) and Drori, Rahimi, et al. (2017). Briefly, frozen leaves were weighed and ground with a pestle. CTAB buffer was added before incubation (65°C, 30 min) in a dry bath and stirred with vortex 3–4 times during incubation. An equal volume of chloroform:isoamyl alcohol (24:1) was added and mixed by inverting tubes. After phase separation, DNA was precipitated by 1/2 volumes of 5 M NaCl and 2 volumes of absolute cold ethanol. The extracted DNA pellets were air‐dried at room temperature and dissolved in 70 µl DNase‐free water (Promega Ltd., USA). Samples were stored in a −20°C freezer until genotyping.

Genotyping of each accession was conducted using a panel of 22 standard SSR markers that were developed for genotyping grapevine (Emanuelli et al., 2013). Two markers (VVMD5 and VVIn73) had high level of missing data and were excluded from the analyses. Multiplex polymerase chain reaction (PCR) amplifications were performed in a final volume of 25 μl containing 50 ng genomic DNA, 12.5 μM Go Taq Green Master Mix (Promega, USA), and 0.4 μM of each primer. The PCR products, including the GeneScan™ 500 ROXW size standard (Thermo Fisher Scientific Ltd., USA), were denatured and size‐fractionated using capillary electrophoresis on an ABI 3500 Genetic Analyzer (Thermo Fisher Scientific Ltd., USA). Finally, allele size estimation at each marker was determined using the software GeneMapper 5 (Thermo Fisher Scientific Ltd., USA).