Dna extraction

DNA extraction , amplification and sequencing

The genomic DNA was isolated from the samples thanks to the Dneasy Plant Mini Kit (Qiagen, CA, USA). Four different molecular markers were amplified to determine the phylogenetic relationship of the taxa within Laserpitium s.l and the Apioideae subfamily sensu Downie et al. (2010): the ribosomal nuclear marker Internal Transcribed Spacer (ITS), the three plastid regions trnL, trnL–F and rpl16 intron. Complete nuclear ITS region (ITS–1, 5.8S and ITS–2) was amplified using the pair of primers N16F and N16R1 of Prieto et al. (2013), modified from Sun et al. (1994), while the plastid regions trnL and trnL–F were amplified with the primers pairs Lc–Ld and Le–Lf respectively ( Taberlet et al. 1991). The plastid rpl16 region was amplified by using the pair of primers F71 ( Jordan et al. 1996) and R1516 ( Kelchner & Clark 1997). The PCR reaction solutions consisted of Standard Reaction Buffer (1x) (Biotools B&M Labs S.A., Madrid, Spain), 1.5 mM MgCl 2, 0.2 mM each dNTPs, 0.8 µM of each primer, 1U DNA polymerase (Biotools), Bovine Serum Albumin (BSA) 1.5mM as PCR enhancer and 10–20 ng of DNA , in a final volume of 25 µl. The PCR cycles involved an initial denaturalization step at 94°C for 5 min followed by 40 cycles of amplification consisting of a denaturalization step at 94°C for 1 min, an annealing step at 54°C for 1 min, and an elongation step at 72°C for 1min, followed by a final elongation step at 72°C for 10 min. Amplicons were sequenced by the Sanger method by Macrogen (Amsterdam, The Netherlands).