DNA was extracted from fruits including the exocarp and the mesocarp. The samples were reduced to a fine powder with liquid nitrogen and DNA was extracted with the QIAGEN DNeasy Plant Mini Kit, following the manufacturer’s instructions. Each sample extraction was repeated three times. After extraction, the concentration values were measured at the Nanodrop. Fourteen primer pairs were used (Table (Table5)5) and were taken from the literature9398. The primers were previously tested on species belonging to the same family, i.e. P. cerasus (sour cherry), P. armeniaca (apricot) and P. persica (peach). SSR markers were selected for their high polymorphism (Table (Table5).5). PCR reactions were prepared in a final volume of 20 µL using genomic DNA at 2 ng/µL, Q5® Hot Start High-Fidelity 2X Master Mix and 5 µM of forward and reverse primers.

Sequences of the 14 primer pairs used for genotyping and relative details. Fluorophore, size range, melting temperature, number of alleles and references are detailed

The PCR parameters were as follows: initial cycle at 98 °C for 30 s, 35 cycles at 98 °C for 10 s, 1 min at 60 °C and 30 s at 72 °C, final extension at 72 °C for 2 min.

PCR products were first visualized on agarose gels. Then, they were multiplexed, diluted in double distilled water (1:50 v/v) and futher analyzed on an ABI3500 Genetic Analyzer (Life Technologies, Waltham, MA, United States). Subsequently, fragment analysis was carried out using the Genemapper 5.0 software. The sweet cherry allelic profiles obtained by genotyping were used to generate a phylogenetic tree using an Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and Sequential Agglomerative Hierarchal Nested (SAHN) cluster analysis with the NTSYSpc software version 2.2 (Exeter Software, USA).

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