2.2 | Amplification of cpDNA and sequencing
PsbA–trnH and rpl14–rpl36 (Sang, Crawford, & Stuessy, 1997) selected from thirteen cpDNA primers were amplified by PCR. PCR were conducted under the following conditions: 94°C (5 min), and 35 cycles at 94°C (30 s) / 56°C (30 s) /72°C (90 s), and then 72°C for 10 min. PCR was performed in a reaction volume of 20 μL containing 2μL of Taq buffer, 20ng of genomic DNA, 1 unit of Taq polymerase, 0.2 μmol/L of each primer and 0.2 mmol/L of each dNTPs. PCR products were checked on 1.5% agarose gels and sequenced at BGI (Beijing, China). Sequences were aligned using CLUSTAL X software (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997).