Dna extraction, , PCR

Panahi, Mehrnoush & Lyskov, Dmitry, 2025, Leutea kurdistanica is a new synonym of Sclerochorton haussknechtii (Apiaceae), a new member of the Cachrys clade, Phytotaxa 693 (1), pp. 55-66 : 56

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from c. 20 mg of dried plant material using DNA extraction kit (Sinaclon Co., Iran) based on CTAB protocol following the manufacturer’s instructions. For PCR amplification of nrDNA ITS and ETS regions, the pair primers ‘N-nc18S10’ and ‘C26A’ ( Wen & Zimmer 1996); and ‘18S-ETS’ and ‘Umb-ETS’ ( Logacheva et al. 2010) were used respectively.

The PCR was performed using ready-to-use MasterMix PCR Kit following the protocol. Details of the PCR ITS and ETS amplification regions were described previously ( Logacheva et al. 2010, Panahi 2023). To check the PCR products, an aliquot of the reaction sample was electrophoresed in a 1% agarose gel based on a TAE buffer and stained with the DNA-safe stain. The successful PCR products were selected and with the same primers were used for subsequent cycle sequencing. Cycle sequencing was performed by automated sequencing using Big Dye terminators (Applied Biosystems-ABI) in the laboratories of MomGene Inc. in Tehran. Both DNA strands across the entire DNA regions were sequenced to avoid ambiguity in base determination. The chromatographs were assembled and edited using SeqMan Pro ver. 12 (Dnastar, Madison, WI, USA). Newly obtained sequences were deposited in the GenBank (Appendix 1).