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Tree Genetics and Molecular Breeding 2012, Vol.2, No.2, 8
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9
rugulosa
, both of them might be the same species. In
addition, the quantitative classification of the genus
Buxus
in China carried out and comparisons with the
morphological classification had done (Yan et al., 2002).
In recent years, molecular analysis is more widely
applied in the phylogenetic study of
Buxus
genus.
Wang et al (2008) analyze isozymes of peroxidase and
esterase and their genetic relationship among three
species in
Buxus
by polyacrylamide gel electrophoresis.
Lv and Ji (2009) carried out fingerprint analysis of
Pearl boxwood clones using isozyme and ISSR. Jiang
(2008) studied the genetic diversity of two natural
populations of Pearl boxwood using RAPD markers.
Huang et al (2008) also conducted genetic structure
and diversity of five natural populations and one
cultivated population of Pearl boxwood using RAPD
and ISSR markers to explore the taxonomical status of
related taxa of the Pearl boxwood.
ITS, inter-transcribed spacer, is located in the middle
between 18S and 26S in rDNA, which was divided
into two parts namely ITS
-
1 and ITS
-
2, by the 5.8S
region. ITS has a feature of conservative in length and
high variability of nucleotide sequence. Therefore,
there are abundant of variable sites and informative
sites. ITS has become an important molecular marker
systems for studying classification and evolution under
the level of genus in angiosperm and has already
applied in phylogenetics in a variety of plant taxa. But
it is yet to be reported genus
Buxus
phylogenetics and
taxonomic status of Pearl boxwood through the ITS
sequences.
In this research, we directly sequenced rDNA ITSs of
five species from Genus
Buxus
and five population of
endangered Chinese Pear boxwood to analyze the
characteristics of ITS and the rules of variation. By
combining the ITS data of other species in
Buxus
deposited in GenBank, we analyzed ITSs of 18 species
in
Buxus
to study the law of molecular evolution as
well as to further understand the classification and
phylogenetic status of Chinese Pear boxwood.
1 Results and Analysis
1.1 Nucleotide substitutions of ITS in
Buxus
plants
ITS sequences of
Pachysandra
terminalis
was used as
the reference sequences, we conducted the alignment
comparisons to account the numbers of nucleotide
transition and transversion (Table 1). The results showed
that in the region of ITS
-
1, the numbers of bases
transiting are almost similar as that of bases transve-
rsing, while in the region of ITS
-
2 the numbers of
base transiting are more than that of base transversing,
this was consistent with the results of Popls plants
(Shi et al., 2006). The same situation took place in the
region of 5.8S but the numbers of five or six nucleotide
substitution appeared only. Little species had obvious
differences reaching ten of nucleotide substitutions or
more. In general,
Buxus
plants had much more numbers
of conversions than that of transversions, and total
numbers of nucleotide substitutions of
Buxus
plants
were far greater than that of
Populus
plants, which
indicated that nucleotide substitutions in the ITS
sequences of the genus
Buxus
should have been
significantly elevated and that nucleotide substitutions
should concentrate in the ITS
-
1 region, accounting for
60% of the total numbers of nucleotide substitution.
1.2 Nucleotide substitution rate of species in
Buxus
Sequence alignment found that informative sites in
ITS of
Buxus
genus was the uneven distribution of
different intervals. Thus, we adopted the Jukes-Cantor
one-parameter model and Kimura's two-parameter
model to analyze the sequences in order to better
reflect the real situation with ITS sequences of
Pachysandra
terminalis
as the reference (Table 2).
By comparison of nucleotide substitution rate, there
was little differences in region of ITS
-
1 between
one-parameter model and two-parameter model, the
average difference between the two sets was 0.0107, P
value of t test was 0.637 (greater than 0.05), which
was not significant. Likewise, the mean difference
between the two sets was 0.0063 in the region of
ITS
-
2, P value of t test was 0.707 (greater than 0.05),
indicating that the results of the two models calculated
the difference should not be significant. Whereas the
average difference between the two sets was 0.0004in
the interval of 5.8S, P value of t test was 0.970
(greater than 0.05), which was not significant. With of
whole ITS region, the average difference of the results
between the one-parameter model and two-parameterm-
odel was 0.0038, being close to the result in the region
of ITS
-
2 due to the significant differences of conversion