Total genomic DNA for all samples was extracted using the standard CTAB
procedure (Doyle & Doyle, 1987) from 30 mg of dried leaf tissue, and
served as the template for the polymerase chain reaction (PCR). DNA
quality and quantity were determined on 0.8% agarose gels stained with
2.5 μL Goldview (Aidlab Biotechnologies Co., Ltd., Beijing, China), with
AL2000 DNA marker (Aidlab Biotechnologies).
One
nuclear ribosomal DNA (nrDNA) sequence, the ribosomal inter-transcribed
spacer (ITS) region comprising spacer 1, the 5.8S ribosomal gene and
spacer 2 (White et al., 1990), and the two chloroplast DNA (cpDNA)
intron-spacer regions trn L-trn F (Taberlet et al., 1991)
and ycf 1b (Dong et al., 2015), were used in this study (Table 2).
PCR reactions were set up in a volume of 25 μL, composed of 20 μL
ddH2O, 2.5 μL 10 × buffer, 0.5 μL 10 mM dNTPs, 0.5 μL
each 5 μM primer, 0.5 μL DNA template and 0.5 μL 5 U/μL Taq polymerase
(Aidlab Biotechnologies). The PCR reactions were carried out on a 2720
Thermal Cycler (Applied Biosystems by Life Technologies, Singapore). The
PCR program for ITS1/2 and trn L-trn F was designed with an
initial denaturation of 5 min at 94 ºC, followed by 35 cycles of 1 min
at 94 ºC, 1 min at 55 ºC, 1 min at 72 ºC, and with a final extension of
10 min at 72 ºC. Amplification of ycf 1b used the following
protocol: 4 min at 94 ºC, 35 cycles of 30 s at 94 ºC, 40 s at 58 ºC, and
1 min at 72 ºC, ending with 10 min at 72 ºC. All the PCR products were
verified based on size by gel electrophoresis. The amplicons were
sequenced by an ABI 3730 DNA Analyzer in forward and reverse directions
based on the BigDye Terminator Cycle Sequencing Ready Kit (Applied
Biosystems, Foster City, CA, U.S.A.) in BGI (Beijing Genomics Institute,
China).
2.2 |
Genetic diversity
The chromatograms from both directions of the ITS1/2 and cpDNA sequences
were checked visually and edited manually with the software BioEdit
(Hall, 1999) for base confirmation and contiguous sequence editing; each
base/character was equally weighted before analysis, and each indel/gap
was represented as a single mutation. Three sequences were manually
aligned and trimmed integrally where necessary using MEGA v.6.5 (Kumar
et al., 2008) separately. The two no-coding cpDNA sequences were
assembled as a single locus for subsequent analysis by SequenceMatrix
v.1.7.8 (Vaidya et al., 2011), and the partition homogeneity test of
cpDNA sequences vs. nrITS sequences was carried out with PAUP* v.4.0a164
(Swofford, 2002). Since non-homogeneity of both matrices was detected,
nrITS and cpDNA were analyzed independently.
DNASP v.6.12.01 (Rozas et al., 2017) was used to compute the number of
identified ribotypes/chlorotypes (Nn ), haplotype diversity
(h ) within populations, polymorphic sites (S ), nucleotide
diversity (π ), and the average number of nucleotide differences
(Nd ) separately for nrITS and cpDNA matrices. The geographic
distribution maps of ribotypes/chlorotypes at the population level, also
separately for nrITS and cpDNA sequences, were constructed and
visualized with ArcGIS v.10.8 (ESRI, Redlands, CA, U.S.A.).