全 文 ：Genetic Diversity of Indocalamus Determined by
Chloroplast DNA Sequence
MU Shao-hua1，QIE Guang-fa2，PENG Zhen-hua2*
1． SFA Key Laboratory of Bamboo and Rattan Science and Technology，International Center for Bamboo and Rattan，Beijing 100102;2． Re-
search Institute of Forestry，Chinese Academy of Forestry，Beijing 100102
Abstract ［Objective］The aim was to research the relationship and genetic diversity of Indocalamus． ［Method］Using 13 samples of Indocala-
mus and 3 samples of Sasa as materials，the intergenic regions of trnL-trnF gene in chloroplast were amplified by PCR，and sequence analysis
and phylogenetic trees construction were carried out． ［Result］Using the universal primer，the intergenic regions of trnL-trnF were amplified，the
lengths of the segments varied from 1 008 bp to 1 103 bp，of which 940 bp was compared． The dendrogram of trnL-trnF sequences showed that
Indocalamus and Sasa were clustered together and they were homologous by 99%． All the samples were divided into five groups，the first group
included 12 samples such as Indosalamus pedalis，I． pumilus，I． victorialis，I． longiauritus，I． tessellatus，Sasa sinica，Sasa pygmaea，I． bar-
batus，I． guangdongensis，I． herklotsii，I． Hirtivaginatus and S． fortunei． I． decorus，I． lacunosus，I． Latifolius and I． Migoi were respectively di-
vided into four groups． ［Conclusion］The high homology of all samples showed the low evolution speed and little information sites which sugges-
ted that the phylogeny of Indocalamus could not be well resolved by the intergenic region of trnL-trnF．
Key words Indocalamus;Chloroplast DNA;Sequence;Diversity
Received:May 26，2010 Accepted:June 9，2010
Supported by:the Supporting Program of the Eleventh Five-year
Plan for Sci ＆ Tech Research (2006BAD19B0202) ;The Program
of Special Funds for basic scientific research of International Center
for Bamboo and Rattan (1632009007) ;Foundation Item of Interna-
tional Center for Bamboo and Rattan (06 /07-C22)．
* Corresponding author． E-mail:mush@icbr． ac． cn
Indocalamus Nakai is a perennial bamboo of Bambuso-
ideae． Because of big leaves，special fragrance，small body
and graceful shape，its considered as a desirable ornamental
and landscaping bamboo． In additional，leaves and bamboo
shoots have medicinal values． Like other bamboos，the flow-
ering period of Indocalamus species is a little long，and mor-
phological classification mainly depends on characteristics of
the trophosome． However，the characteristics of the tropho-
some may usually generate some variances within a great
range due to the environmental effect［1］，which brings some
difficulties to classify and study this genus．
Chloroplast DNA (cpDNA)is maternally inherited． Com-
pared with nuclear genome，its molecular weight is smaller，
copy number is larger，and molecular structure is simp-
ler［2 －4］． The homology analysis of cpDNA has proved to be an
important method to research affinity of plants owing to its
conservatism and monolepsis． The intron sequence of trn in
cpDNA genome is more widely used as one of molecular tar-
gets in molecular systematics studies，because the extent of
sequence variation is not conservative(compared with the se-
quence of mitochondrial gene) ，neither not significant (com-
pared with nuclear genome)． The intergenic non-coding re-
gion of trnL(UAA)-trnF(GAA)is the most common non-cod-
ing region of cpDNA during the research of phylogeny［5 －8］．
The length of trnL-trnF gene sequence is not the same in dif-
ferent groups． And not restricted by functions，the evolution-
ary rate is greater than that of functional coding region． Fur-
thermore，there are many insertions /deletions and high pro-
portion of substitution variation． All these make the intergenic
non-coding region of trnL(UAA)-trnF(GAA)gene suit to
solve some problems about the evolution of the related spe-
cies and even sub species［9］．
In order to provide a basis for further verifying whether
one cpDNA gene fragment can illuminate the systematics
classification of Indocalamus，the authors selected the inter-
genic regions of trnL-trnF gene in chloroplast，and took ac-
tions of genetic manipulation and sequence analysis on cpD-
NA genome of 13 samples of Indocalamus and 3 samples of
Sasa． What had done was to preliminarily reveal their affinity，
and to provide references for germplasm resource preserva-
tion and sustainable utilization of this genus．
Materials and Methods
Materials
After the collected 13 species of Indocalamus and 3 spe-
cies of Sasa (showed in table 1)were identified by experts，
20 plants were randomly selected from each species，and one
tender fully expanded leaf was selected from each plant．
Then，the same amount of mixed samples was taken out as a
sample for total DNA extraction．
Table 1 The sources and codes of bamboo species
Code Scientific name Sources of meterials
1 Indocalamus decorus Bamboo species garden ofZhejiang Forestry Academy
2 I． lacunosus Bamboo species garden ofZhejiang Forestry Academy
3 I． Latifolius Bamboo species garden ofZhejiang Forestry Academy
4 I． migoi Bamboo species garden ofZhejiang Forestry Academy
5 I． pedalis Bamboo species garden ofZhejiang Forestry Academy
6 I． pumilus Bamboo species garden ofZhejiang Forestry Academy
7 I． victorialis Bamboo species garden ofZhejiang Forestry Academy
8 I． longiauritus Century bamboo garden inChangning，Sichuan
9 I． tessellatus Century bamboo garden in-Changning，Sichuan
Agricultural Biotechnology
Agricultural Science ＆ Technology，2010，11(4):50 －52，89
Copyright 2010，Information Institute of HAAS． All rights reserved．
DOI:10.16175/j.cnki.1009-4229.2010.04.040
Continued
Code Scientific name Sources of meterials
10 Sasa sinica Century bamboo garden inChangning，Sichuan
11 Sasa pygmaea Bamboo species garden ofNanjing Forestry University
12 I． barbatus Resource garden of taipingbase in Huangshan
13 I． guadongensis Resource garden of taipingbase in Huangshan
14 I． herklotsii Resource garden of taipingbase in Huangshan
15 I． Hirtivaginatus Resource garden of taipin basein Huangshan
16 Sasa fortunei Resource garden of taipingbase in Huangshan
The primers，c and f，were designed by Beijing branch of
BIOASIA，and were diluted to 20 pmol /μl． Taq DNA polymer-
ase was bought from TaKaRa． PCR amplification and product
purification were accomplished in SFA Key Laboratory of
Bamboo and Rattan Science and Technology，and sequen-
cing was finished by Beijing Biological Technology Company．
Methods
DNA extraction The total DNA was extracted by CTAB
method［10］． The content of DNA was detected by agarose gel
electrophoresis． Then，the qualified DNA samples were dilu-
ted to 50 －100 ng/μl，and ready to be used．
The DNA fragments amplification 1 μl of the DNA sample
referred above，1 μl of each primer，37．75 μl of ultrapure wa-
ter，4 μl of dNTP Mixture (2． 5 mmol /L) ，5 μl of 10 × PCR
Buffer (Mg2 + Plus)and 0． 25 μl of Taq DNA polymerase (5
U/μl)was added into a centrifuge tube． The process of ampli-
fication was performed with PCR instrument (Biometra Tgra-
dient)according to Lu Mengzhus method． The size and con-
centration of amplified products were determined by agarose
gel electrophoresis (1． 0%)［3］． Results were observed and
taken photos by Ultraviolet Detector (VilBer lourmat)．
Amplified products purification Protoal method of the
Winzard PCR Preps DNA Purification System from Promega
was used to purify DNA，which was conserved at －20 ℃．
Sequence determination The purified products were se-
quenced by Beijing Biological Technology Company． And the
type of automatic sequencer is ABI3730XL．
Sequence analysis and phylogenetic trees construction
Sequences were aligned in Clustal X［11］，and manually adjus-
ted． Primers and partial sequence on the edge were deleted，
and the two-dimensional determined sequence was spliced in-
to a complete sequence in accordance with the normal direc-
tion． Afterwards the collated sequence was to be analyzed the
phylogenetic development and homology by DNAMAN (ver-
sion 4． 0，Lynnon Biosoft 1994 －1998)． Bootstrap(1 000 rep-
etitions)［12］ was used to inspect the confidence coefficient of
each branch．
Results and Analysis
trnL-trnF gene fragments amplification
The length of trnL-trnF gene fragments is nearly 1 000
bp，which were respectively amplified from sixteen bamboo
samples by PCR，using the reported universal specific primer．
The trnL-trnF gene fragment sequence mutation
16 trnL-trnF gene fragment sequences were obtained af-
ter sequenced． The lengths of the segments varied from 1 008
bp to 1 103 bp，of which 940 bp was compared． Thats to
say，their comparability is 99． 52%． The variances between
samples were mainly the base deletion，A and G or A and C
conversion，etc．
Phylogenetic location analysis of Indocalamus
The genetic distance between these 16 species was cal-
culated based on cpDNA sequences noted above． The phylo-
genetic location was analyzed by DNAMAN (version 4． 0) ，
and the homology-tree was reconstructed (Fig． 1)．
Fig． 1 The homology-tree reconstructed for Indocalamus
based on the sequence of trnL-trnF intergenic region
The dendrogram of trnL-trnF sequences showed that In-
docalamus and Sasa were clustered together and they were
homologous by 99%，which can be seen from Fig． 1． All the
samples were divided into five groups． The first group includ-
ed 12 samples such as Indosalamus pedalis，I． pumilus，
I． victorialis，I． longiauritus，I． tessellatus，Sasa sinica，Sasa
pygmaea， I． barbatus， I． guangdongensis， I． herklotsii，
I． Hirtivaginatus and S． fortunei，which were high homolo-
gous． I． decorus，I． lacunosus，I． Latifolius and I． Migoi were
respectively divided into four groups．
Discussions
Genetic relationship of Indocalamus
Since the flowering period of most bamboos is long，it is
not easy to obtain flowers and fruits． Therefore，morphologi-
cal classification mainly depends on characteristics of the tro-
phosome． As a result，the bamboo becomes one of the cha-
otic groups in the classification of angiosperm． As a kind of
temperate woody bamboos，the administrative relationship of
many species of this genus is still confusion，and the number
of this genus is inconsistent，because no type specie of this
genus was specified at that time． Neither，the existing mor-
phology-based classification system is mostly unnatural，and
its own scientificity needs to be verified［13］．
The trophosomes shape is much similar between bam-
boos of Indocalamus and S． fortunei，I． Hirtivaginatus and
I． longiauritus of Sasa． Sasa is unusually considered as one
of bamboo species of high altitude，but Indocalamus is one of
bamboo species of low altitude． Besides，the number of an-
droecium and the shape of vascular bundle of the two genera
are different［14］． The species between Indocalamus and Sasa
were knotted together，and the internal branching didnt tally
with the division of the traditional classification． These two
genera should be merged as the clustering result showed，
which didnt support the existing morphological classification．
These bamboos are extremely high homologous，espe-
cially the twelve samples in group 2． They are homologous by
about 100%． The relationship among species is not indeci-
sive，so it is not suitable to discuss the classification． If further
discussion will be done，morphologic and more convincing evi-
15MU Shao-hua et al． Genetic Diversity of Indocalamus Determined by Chloroplast DNA Sequence
dence of molecular biology are needed．
The analysis of the system location based on sequence of
cpDNA gene
The intergenic non-coding region of trnL (UAA)-trnF
(GAA)gene is the most common non-coding region of cpD-
NA in the study of phylogeny． Smaller molecular weight，lar-
ger copy number，and simpler molecular structure，all these
are propitious to do genetic manipulation and analysis on cpD-
NA genome． The intergenic regions of trnL-trnF gene in chlo-
roplast were non-coding regions，so they withstood less pres-
sure of natural selection and the generated mutation had a
great chance to be reserved． Such extent of mutation can
provide a better resolving power during the analysis of system
location，which is suited to clarify the relationship between
species and ensure the accuracy of system location analy-
sis［15］． Lu Mengzhu revealed the affinity of different geograph-
ical varieties of diversifolious poplar (Populus euphratica Oli-
ver)based on the spacer DNA analysis，in order to lay the
foundation for establishing a complete theory of its systematic
development［3］． Compared the polymorphism of cpDNA，
Tang Qian analyzed the cpDNA differentiation and the muta-
tion extent of larch (Larix gmelinii Rupr)and the phylogenetic
relationship between its species． From a fresh perspective，
he disclosed the phylogenetic relationship between spe-
cies［16］． The intergenic regions of trnL-trnF gene in chloroplast
is steady，and dont change with the seasons changing．
Whereas its only a part of DNA，its not enough to explain all
the problems of systematic development［17］． Besides，the a-
mount of cumulate variance of this gene is not the same to dif-
ferent plant groups．
It was very tough to gain a definite branch result during
the research of molecular systematics on Bambuso-
ideae［18 －20］，which caused some difficulties to system recon-
struction and classification． Investigating reasons，the most
likely one is the long lifecycle of bamboos． Usually their lifecy-
cle is more than five to thirty years，so the chance of gene re-
combination is less． Therefore，the molecular evolutionary
rate is slower than that of other plants． Such problem existed
in this study． The intergenic regions of trnL-trnF gene in chlo-
roplast are considered greater for molecular marker to re-
search the affinity between species in angiosperm． So the au-
thors once tried to resolve the system location of this group
with such gene． However，they found genetic variation was
very little after sequence comparison． What showed was slo-
wer evolutionary rate and provided was fewer informative
sites，which reduced the reliability when constructing the mo-
lecular systematic tree． The result of this DNA analysis pro-
vided didnt support that Indocalamus and Sasa exist sepa-
rately． This result and the current classification，as well as the
result of AFLP molecular marker were both significantly differ-
ent［21］． Sequentially，its difficult to draw a definite conclusion．
Due to the low evolutionary rate (the variation between differ-
ent species is less than 1%) ，this gene fragment in chloro-
plast cant provide essential information of systematic develop-
ment． As a result，it cant clearly demonstrate the systematics
issue between species in Indocalamus．
References
［1］YANG GY(杨光耀)，ZHAO QS(赵奇僧)． Phylogenetic relation-
ships among five Pleioblastus species(Bambusoideae)from China
and Japan based on random amplified polymorphic DNA(苦竹类植
物RAPD分析及其系统学意义)［J］． Acta Agriculturae Universitatis
Jiangxiensis(西北农业大学学报)，2000，22(4):551 －553．
［2］HUANG Y(黄瑶)． Chlohoplast DNA and its application to plant sys-
tematic studies(叶绿体 DNA及其在植物系统学研究中的应用)［J］．
Chinese Bulletin of Botany(植物学通报)，1994，11(2):11 －25．
［3］LU MZ(卢孟柱)，XIE HL(谢红丽)，ZHANG H(张辉)，et al． Study
on phylogeny of Populus euphratica by the chloroplast DNA varia-
tion(利用叶绿体 DNA变异研究胡杨系统发育)［J］． Acta Botanica
Boreali-Occidentalia Sinica(西北植物学报)，2000，20(6):1148 －
1154．
［4］TANG LH(汤陵华)，SUN JX(孙加祥)，UDATSU T(宇田津撤朗)，et
al． Comparison of different classifying methods between indica-type
rice and japonica-type rice on land race rice in Taihu Lake district(太
湖地区水稻地方品种籼粳分类方法的比较)［J］． Jiangsu Journal of
Agricultural Sciences(江苏农业学报)，2003，19(3):139 －144．
［5］BOHE UR，HILGER H，CERFF R，et al． Noncoding chloroplast
DNA for plant molecular systematics at the infrageneric level［J］．
EXS，1994，69:391 －403．
［6］GIELLY L，TABERLET P． The use of chloroplast DNA to resolve
plant phylogenies:noncoding versus rbcL sequences［J］． Molecu-
lar Biology and evolution，1994，11:769 －777．
［7］HAM RCHJ，HART HT，MES THM，et al． Molecular evolution of
noncoding regions of the chloroplast genome in the Crassulaceae
and related species［J］． Current Genetics，1994，25:558 －566．
［8］MES THM，HART HT． Sedum surculosum and S． jaccardianum
(Crassulaceae)share a unique 70 bp deletion in the chloroplast
DNA trnL(UAA)-trnF(GAA)intergenic spacer［J］． Plant Systemat-
ics and Evolution，1994，193:213 －221．
［9］CURTIS SE，CLEGG MT． Molecular evolution of chloroplast DNA
sequences［J］． Mol Biol Evol，1984，1:291 －301．
［10］THOMPSON JK，HIGGINS DG，GIBSON TJ． Clustal W:improving
the sensitivity of progressive multiple sequence alignment through
sequence weighting，positions-specific gap penalties and weight ma-
trix choice［J］． Nucleic Acids Research，1994，2:4673 －4680．
［11］THOMPSON JD，GIBSON TJ，PLEWNIAK F，et al． The Clustal-X
windows interface:flexible strategies for multiple sequence align-
ment aided by quality analysis tools［J］． Nucleic Acids Research，
1997，25:4876 －4882．
［12］FELSENSTEIN J． Confidence limits on phylogenies:an approach
using the bootstrap［J］． Evolution，1985，39:783 －791．
［13］BOHS L，OLMSTEAD RG． Phylogenetic relationships in Solanum
(Solanaceae)based on ndhF sequences［J］． Systematic Bota-
ny，1997，22:5 －17．
［14］WEN TH(温太辉)． Some ideas on the taxonomy of several Bam-
busoideae taxonss(关于几个竹亚科分类群的分类问题)［J］． Jour-
nal of Bamboo Research(竹子研究汇刊)，1991，10(1):11 －25．
［15］DEMESORE B，SODZI N，PETIT RP． A set of universal primers
for amplification of polymorphic non-coding regions of mitochondri-
al and chloroplast DNA in plants［J］． Mol Eol，1995，4:129 －131．
［16］TANG Q(唐谦)，GU WC(顾万春)． The differentiation of chloro-
plast DNA among several larch species and its implications in phy-
logenetic relationship(几种落叶松树种叶绿体 DNA的分化及其系
统演化关系的含义) ［J］． Scientia Silvae Sinicae(林业科学)，
1995，31(4):373 －379．
［17］TIAN X(田欣)，LI DZ(李德铢)． Application of DNA sequences in
plant phylogenetic study (DNA序列在植物系统学研究中的应用)
［J］． Acta Botanica Yunnanica(云南植物研究)，2002，24(2):170
－184．
［18］ ZHANG W． Phylogenetic and classification of the bamboos
(Poaceae:Bambusoidea)based on molecular and morphological
data［D］． Ames:Iowa State University，1996．
［19］HODKINSON TR，RENVOIZE SA，CHONGHAILE GN，et al． A
comparison of ITS nuclear rDNA sequence data and AFLP mark-
ers for phylogenetic studies in Phyllostachys (Bambusoideae，
Poaceae) ［J］． J Plant Res，2000，113:259 －269．
［20］GUO ZH，CHEN YY，LI DZ，et al． Genetic variation and evolution
of the alpine bamboos(Poaceae:Bambusoideae)using DNA se-
quence data［J］． Journal of Plant Research，2001，114:315 －
322．
［21］MOU SH(牟少华)，PENG ZH(彭镇华)，SUN QX(孙启祥)，et al．
Genetic diversity of Indocalamus determined by AFLP marker(部
分箬竹属植物的荧光 AFLP分析)［J］． Scientia Silvae Sinicae(林
业科学)，2009，45(11):32 －35．
Responsible editor:KONG Qiu-sheng Responsible proofreader:WU Xiao-yan
(下转第 89 页)
25 Agricultural Science ＆ Technology Vol． 11，No．4，2010
［6］XUE RC(薛永常)，NIE HZ(聂会忠)，LIU CB(刘长斌)． Bioinfor-
matics analysis on C3H in different plants(木质素合成酶 C3H基因
的生物信息学分析)［J］． China Journal of Bioinformatics(生物信息
学)，2009，7(1):13 －17．
［7］KAGEYAMA H，ISHII A，MATSUOKA T，et al． Simple isolation of
phycocyanin from Spirulina platensis and phycocyanobilin-protein
interaction［J］． Mar Biotechnol，1994，1(4):185 －188．
Responsible editor:QIAO Li-li Responsible proofreader:
檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
WU Xiao-yan
极大螺旋藻藻胆蛋白 α亚基基因的生物信息学分析(摘要)
刘士伟1，刘 杰2，李博生1* (1．北京林业大学生物科学与技术学院，北京 100083;2．北京林业大学螺旋藻研究所，北京 100083)
［目的］对极大螺旋藻藻胆蛋白 α亚基进行分析研究，为该蛋白的后续研究提供依据。
［方法］用生物信息学分析方法对已经在 GenBank上登录的极大螺旋藻藻胆蛋白的 α亚基基因(GenBank AF441177)及其翻译的氨基酸序列
进行了其组成和相关性质的分析、蛋白质信号肽预测、跨膜结构域的预测及亲水性 /疏水性的测定，同时构建了分子进化树，对上述结果进行
了分析和探讨。
［结果］该蛋白的氨基酸组成丰富，不仅含 18种必需氨基酸还含有含有甘氨酸、天冬氨酸等非必需氨基酸;对该蛋白信号肽和跨膜结构域分
析表明，其属于胞内蛋白;对该蛋白的亲水性分析表明，其属于亲水性蛋白;进化树分析表明，该蛋白与节旋藻的同源性高，达 99% ～100%。
［结论］该研究为 α亚基和 β亚基之间的关系和相互作用提供了一定依据。
关键词 藻胆蛋白;α亚基;生物信息学
基金项目 国家林业公益性行业科研专项资助(200704025);教育部重点研究项目(102023)。
作者简介 刘士伟(1983-)，男，山东临清人，硕士研究生，研究方向:螺旋藻开发利用。* 通讯作者。E-mail:lbs7778@ yeah． net
收稿日期 2010-05-18 修回日期
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2010-06-23
(上接第 52页)
部分箬竹属植物的叶绿体 DNA分析(摘要)
牟少华1，郄光发2，彭镇华2* (1．国际竹藤网络中心，国家林业局竹藤科学与技术重点开放实验室，北京 100102;2．中国林业科学研究院
林业研究所，北京 100102)
［目的］研究箬竹属植物和赤族属植物的亲缘关系。
［方法］以箬竹属 13个竹种及其近缘的赤竹属 3个竹种为材料，采用 PCR方法扩增叶绿体 trnL-trnF间隔区基因片段，并对其进行序列分析，
构建系统树。
［结果］利用已发表的 trnL-trnF序列通用引物扩增出长度为 1 008 ～1 103 bp的 trnL-trnF片段，比较长度为 940 bp。cpDNA序列聚类将箬竹
属与赤竹属竹种混合聚在一起，同源性为 99%以上，可分为五组。其中，髯毛箬竹、广东箬竹、小叶箬竹、华箬竹、毛鞘箬竹、矮箬竹、胜利箬
竹、箬竹、箬叶竹、粽巴箬竹、翠竹和菲白竹 12个竹种聚为一组;泡箬竹、天目箬竹、阔叶箬竹和美丽箬竹 4个竹种各自成一组。
［结论］竹种间的同源性极高表现为较慢的进化速率，提供的信息位点很少，不能很好地解决箬竹属种间的系统学问题。
关键词 箬竹属;叶绿体 DNA;序列分析;多样性
基金项目 “十一五”科技支撑项目(2006BAD19B0202);国际竹藤网络中心基本科研业务专项资金项目(1632009007);国际竹藤网络中心基金项目
(06 /07-C22)。
作者简介 牟少华(1976 －)，女，山东栖霞人，博士，助理研究员，从事种质资源和分子生物学方面的研究。* 通讯作者。
收稿日期 2010-05-26 修回日期 2010-06-09
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