DNA extraction and microsatellites amplification

Genomic DNA samples were extracted from silica gel dried leaves using the GenElute^™ Plant Genomic DNA Miniprep Kit (Sigma-Aldrich^®). Eight highly informative microsatellite markers were used for the analysis: SoUZ001, SoUZ002, SoUZ003, SoUZ007, SoUZ011 [62], and SoUZ013, SoUZ014, and SoUZ019 [63]. PCR amplification reactions were performed in a total volume of 20 μL containing, 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 U of Taq HS polymerase (Takara Bio Inc.), 0.2 μM of fluorescent labelled forward primer (FAM, VIC, NED or PET), 0.2 μM of reverse primer (Applied Biosystems^®) and 5 ng of genomic DNA. DNA amplification was performed using a GeneAmp PCR System 9700 (Applied Biosystems^®) and a two-step PCR protocol with an initial touchdown cycle. The cycling conditions were as follows: 94°C for 5 min; 5 cycles of 45 s at 94°C, 30 s at 60°C, which was lowered by 1°C in each cycle, and 90 s at 72°C; 25 cycles of 45 s at 94°C, 30 s at 55°C, and 90 s at 72°C; and an 8 min extension step at 72°C. The products were run on an ABI 3730XL (Applied Biosystems^®) analyser using the commercial GeneScan service (Macrogen Inc.). The results were analysed using GeneMapper 4.0 software (Applied Biosystems^®).