Genemapper genotyping software v 3

A set of 12 microsatellite markers, previously proven to be highly performant for genetic olive characterization, were used (Table S7) [54 (link),55 (link),56 (link)]. PCR reactions were conducted in a final volume of 12.5 µL, according to di Rienzo et al. [6 (link)]. In brief, 1.25 µL of 10X Dream Taq Buffer, 0.6 µL of 2M dNTP, 1.25 µL of a mix of primers (2.5 µM), 0.2 µL of Dream Taq, and 7.7 µL H₂O were added in each well containing 50 ng of DNA. PCR amplifications were performed in a C1000TM Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the products were checked in 1.5% agarose gel. PCR products were detected by the automatic capillary sequencer ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with the internal molecular weight standard GeneScan Liz 600 dye (Applied Biosystems, Foster City, CA, USA). GeneMapper genotyping software v.3.7 (Applied Biosystems, Foster City, CA, USA) was used in order to carry out the sample analyses.

A set of 11 microsatellite markers [simple sequence repeat (SSR)] was selected as the most effective in differentiating the olive accessions (Table S1) (Boucheffa et al., 2017 (link)). PCR reactions were performed in a C1000 Touch^TM Thermal Cycler (Bio-Rad, Hercules, CA, USA) following the protocol described in Montemurro et al. (2015) (link). In order to verify PCR efficiency, PCR products for each of the 11 SSR markers were randomly checked by electrophoresis on 2.5% SeaKem LE Agarose gel (Lonza, Visp, Switzerland). The amplification products were detected by the automatic sequencer ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and the sample analyses were carried out using the GeneMapper genotyping software v3.7 (Applied Biosystems, Foster City, CA, USA). The internal molecular weight standard was GeneScan^TM 500 ROX dye Size Standard (Applied Biosystems, Foster City, CA, USA).