DNA extraction, PCR amplification, and sequencing
The genomic DNA was extracted by CTAB-SiO2 method (Nan,
2012; Zhang et al, 2004). 3 chloroplast DNA (cpDNA) fragments, selected
after screening: matK, trnD-E, trnS-G, and the nuclear internal
transcribed spacer (ITS) were amplified. Polymerase chain reaction (PCR)
amplifications (25 μL) contained: 12.5 μL 2 × Taq PCR MasterMix
(Tiangen, Beijing, China), 1 μL each primer, 2 μL DNA template and 8.5
μl ddH2O. The PCR procedure was as follows: initial
denaturation (95 ◦C, 5 min), 35 cycles of denaturation (94 ◦C, 45 s),
annealing (52/56 ◦C, 45 s) and extension (72 ◦C, 45 s), and a final
extension (72 ◦C, 8 min). The PCR products were checked by a 1.0%
agarose gel and were sent for sequencing on ABI Prism 3730 Genetic
Analyzer (Sangon, Shanghai, China).