Material collection was approved by the Consejería de Educación e Investigación de la Comunidad de Madrid (REMEDINAL-TE CM, P2018/EMT-4338). The genetic study was performed with eight specific microsatellites markers: Lsub01, Lsub02, Lsub03, Lsub04, Lsub05, Lsub07, Lsub08, Lsub12 (Martínez-Nieto et al., 2012). DNA was extracted from 60 mg of dried young leaf tissues using the SpeedTools Plant Extraction kit (Biotools, Madrid, Spain). We amplified individually each microsatellite using Polymerase Chain Reactions, mixing 14.2 μl of milli-Q water, 2 μl of T10X buffer with MgCl2, 0.8 μl of dNTP mix [10 mM], 0.5 pmol of each primer (forward labelled with fluorescence dye and reverse) 1 U of Taq-polymerase (Biotools, Madrid, Spain) and 1.2 μl of DNA (DNA had a concentration range of 10–30 ng/ul). The thermocycler’s protocol was: 4 min at 95 °C, followed by 30 cycles of 45 s at 95 °C, 45 s at 51 °C (annealing temperature) and 1 min at 72 °C, followed by a final step of 7 min at 72 °C. PCRs were checked using electrophoresis in agarose gel pre-stained with Gel Red (Biotium, Hayward, CA, USA). Lastly, the amplified fragments were analysed with an ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA) of the Unidad de Genómica (Universidad Complutense, Madrid, Spain).
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