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Tree Genetics and Molecular Breeding 2012, Vol.2, No.2, 8
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14
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13
hypothesis (Luo and Zhang, 2000). In this study, The
results showed that there was a greater difference in
the rate of nucleotide substitution of ITS gene in the
genus
Buxus
as well as a significant difference in the
rate of evolution among cluster groups, so this would
provide new evidence not only for the molecular
allometric phenomenon in the evolutionary process,
but also for in-depth understanding phylogenetic
relationships the genus
Buxus
.
3 Materials and Methods
3.1 Experimental materials
In this study, five species of
Buxus
genus widely
distributed in China, including five populations of
pearl boxwood (four wild species, one cultivated
species) were used for molecular evolution studies. In
addition, nine ITS sequences of
Buxus
plants were
downloaded from GenBank, and reference sequence
was
Pachysandra
terminalis
that has close genetic
relationship with the
Buxus
plants (Table 4).
3.2 DNA extraction
Using a modified CTAB method, we extracted
genomic DNA of
Buxus
species following with the
reference of Lv et al (2007).
3.3 ITS sequences amplified by PCR
Using 50 μL PCR reaction system including the
double-tranded DNA template 50~100 ng, MgCl
2
(25 mmol/L), 4 μL, dNTP (2.5 mmol/L), 6 μL,
Taq
DNA polymerase (TaKaRa) 1.75 U, forward primer
0.75 μL (5'
-
AGAAGTCGTAACAAGGTTTCCGTAG
G
-
3', reverse primer 20 μmol/L, 5'
-
TCCTCCGCT
TATTGATATG C
-
3', 20 μmol/L), and adding ddH
2
O
to 50μL. PCR amplification procedures as 94
℃
pre-denaturation 4 min, and then 30 cycles with 94
℃
denaturation for 30 s, 57
℃
annealing for 30 s, and 72
℃
extension for 1 min, finally, after 72
℃
extension 7 min,
placed at 4
℃
ready for use. PCR products were
purified by using purification kit (AxyPrep DNA Gel
Extraction Kit) to be as sequencing template for
sequencing directly.
3.4 ITS sequencing
Sequencing was performed by Shanghai Yinjun
Biotechnology Company. Adopting two of four classic
sequencing primers in the 5.8S reported previously,
P2: (5'
-
GCATCGATGAAGAACGCAGC
-
3') and P3
(5'
-
GCTGCGTTCTTCATCGATGC
-
3') (Wang et al.,
1999) for the sequencing primers, synthesized by
Shanghai Yinjun Biotechnology Company.
Table 4 The materials used in the research and their sources
No. Name
Source
No. Name
Source
1
Buxus sinica
var.
parvifolia
Qingliangfeng Anhui(wild)
11
Buxus macowanii
Genbank
AF245411
2
Buxus sinica
var.
parvifolia
Wanfoshan Anhui(wild)
12
Buxus microphylla
var.
japonica
Genbank
AF245412
3
Buxus sinica
var.
parvifolia
Yaoluoping Anhui(wild)
13
Buxus riparia
Genbank
AF245413
4
Buxus sinica
var.
parvifolia
Tianzhushan Anhui (wild)
14
Buxus balearica
Genbank
AF245423
5
Buxus sinica
var.
parvifolia
Rugao Jiangsu(cultivated)
15
Buxus glomerata
Genbank
AF245426
6
Buxus sempervirens
Wuyishan Fujian
16
Buxus gonoclada
Genbank
AF245427
7
Buxus sinica
Nanjing Forestry University
17
Buxus liukiuensis
Genbank
AF245428
8
Buxus henryi
Zhongshan botanical garden Nanjing 18
Buxus citrifolia
Genbank
AF245433
9
Buxus bodinieri
Zhongshan botanical garden Nanjing 19
Pachysandra terminalis*
Genbank
AF245430
10
Buxus harlandii
Genbank
AF245410