全 文 ：广 西 植 物 Guihaia 25(2)：129— 133 2005年 3月
利用RAPD研究桂林桂花品种间的亲缘关系
伊艳杰1，黄 莹2，尚富德
(1．河南大学生命科学学院 ，河南开封 475001；2．桂林黑山植物园，广西桂林 541002)
摘 要：采用随机扩增多态性 DNA(RAPD)技术，从 100个随机引物中筛选出扩增效果较好的2o个引物，分
析桂林市 23个桂花品种的基因组多态性。2O个随机引物共检测到 193个位点，其中多态位点 114个，占
59．1％。并进行了聚类分析，构建出树状聚类图，将这些品种划分为 4个品种群，与传统分类学结果一致。结
果表明，以基因型而不是以表现型为基础，分析桂花品种问的区别是可能的。该技术为解决桂林市的桂花品
种分类问题提供了重要依据。
关键词：RAPD；桂林；桂花；亲缘关系
中图分类号：$685．1 3 文献标识码：A 文章编号：1000—31 42(2005)02 0129 05
Identifying Osmanthus fragrans cultivars
Guilin City and evaluating their genetic
relationships by RAPD markers
YI Yan-jie ．I{UANG Ying ，SHANG Fu—de
(1．College of Li，e Sciences，Henan University，Kaifeng 475001，China；
2．Hei5tzan Botanica Garde ．G uilin 54 1002，China)
Abstract：Random amplified polymorphic DNA(RAPD)markers were applied to analyze 23 Osmanthus fra—
gra~s cuhivars in Guilin City．Among the total 193 fragments generated by 20 selected primers(among 100
primers)，1 14 appeared to be polymorphic(59．1 )．Cluster analysis based on the RAPD results was per—
formed and dendrogram was constructed．These cuhivars tested by RAPD were divided into 4 cuhivar groups．
The result was consistent with that from traditional taxonomy analysis．The RAPD study ilustrated that it is
possible tO analyze intra～cuhivar variation of O．fragrans cuhivars on the basis of genotype rather than pheno—
type and therefore，offered significant evidences in solving taxonomic problem of O．fragrans cuhivars in Ouil—
in City．
Key words：RAPD；Guilin；Oslnant]zus
．
fragrans；genetic relationship
Osmanth HS fragrans originated from China
and belonged to Osmanthus．As one of ten tradi—
tional famous flowers in China，O．．厂’ragrans is well
known because of its sweet smel1(Liu et a1．。
2000)．It iS widely cultured in middle and northern
semitropical areas．Guilin City is rich in O．fr．一
grans(Yang et a1．，2000)．However，there is insuf—
ficient study on abundant resources of 0．fragrans
cuhivars in Guilin City．In China，O．fragrans had
been studied since 1940s(Huang et a1．，1949)and
played an important role in flower culture．In the
past，classical approaches for the identification to
收稿日期：2004—02～09 修订日期：2004～05—2O
基金项目：河南省自然科学基金(0311031400)
作者简介：伊艳杰(1978一)，女，河南许昌人，博士研究生．从事植物分子系统学研究。 通讯作者E-mail：fudeshang’@henu．edu．cn
n
●l
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l3O 广 西 植 物 25卷
0．fragran cuhivars were based on morphological
and Dhysio1ogical traits．Most morphological traits
are easilv affected by environmental factors．So it
is difficuh to assess these traits and their evalua—
tion can be subjective considering that most of
these cuhivars are related(Liu，2000；Obara—Okeyo
et a1．，l998)．
In 1995，the use of isozyme analysis to identify
O．fragrans cuhivars in Henan Province was re—
Dorted(Chen e￡“￡．，1995)． But the ability of
isozymes to identify cultivars was limited due to
1ack of sufficient polymorphism．Recently，the ran—
dom amplified polymorphic DNA technique(W elsh
￡“Z．，l990；Williams et al1．，l990)based on the
polymerase chain reaction(PCR)has been widely
used for cultivar identification．The basis o{genet—
ic variability is sequence va riation．RAPD are mo—
lecu1ar markers that samples and reveals sequence
variati0n bv differential amplification of DNA frag—
ments(Zhu et“￡．，l999)．There was only one re—
Dort on RAPD analysis in O．frag7 arls colected
from W uhan City(Zhao et“Z．，1999)，which illus—
trated that genetic diversit 3，between O．fragrans
cultivars cou1d be measured as RAPD markers di—
versity。Our objective was 1 0 analyze genetic rela—
tionships among O．fragrans cuhivars by RAPD
technique and classify these cuhivars based on
RAPD markers．The study of genetic diversity a—
mona cuhivars will be of significance in germplasm
protection and conservation．
1 Materials and methods
1．1 Plant materials
23 0． “ Tails cuhivars were all collected
from Heishan Botanical OaI·den in Guilin City of
Guangxi Province． Leaves of these cuhivars were
dried by Silica gel and put in refrigerator．These
cuhivars were：(1)0．fragran s‘Yuanban Jingui’；
(2)O．fragrans‘Taoye Jingui’；(3)0．fragrans
‘Xiangjingui’：(4)0．frag ‘Jinlian’；(5)0．
fragrans‘Zi’e’；(6)0．．厂，-“grans‘Mantianxing’；
(7)0．fragtans‘Nongchao- r’；(8)0．fragrans
。Qingyun’；(9)0．fragrans‘YaotiaoshuntX’；(i0)
0．fr“g，-c￡n ‘DayeYingui’；(1 1)0．
．
fragrans‘Xi—
aove Yingui’；(12)0．fragran s‘Ruichi Yingui’；
(13)0．fr“grans‘Zi Yingui’；(14)0．fragrans
c Ruichi Ziyingui’；(15)0．fragrans‘Taoye Ziyin—
gui ；(16)0．fragrans‘Xiaoye Ziyingui’；(17)O．
f，-“ ，-(￡ ‘Meixin’；(18)0．
．
fragrans‘Nanxi Dan—
gu (19)0．fragrans‘Guifeihong’；(20)O． “一
譬r“ ‘Zidangui’；(21)0．fragrans‘Danxin’；(22)
0．fragran s‘SiSigui’；(23)0．．fragrans‘Yueyue—
gut ．
1．2 DNA extraction
DNA was extracted from 0．5 g of cold and dry
leaf．Leaf tissue was ground to a fine powder in
liquid nitrogen．and then placed in a 10 mL eppen—
dorf tube．3 mL 2 X CTAB extraction buffer pre—
heated was added to the tube，mixed gently by in
version and incubate at 65℃ in a hot water—bath
for 3O min．Then l mL(1／3 volume)KAc was add—
ed and the eppendorf tube was cooled immediately
in ice water for 2O min． An equal volume of 24
chloroform：1 isoamyl alcohol(v／v)was used for
extraction for l 0 min with gentlly but thoroughly
mixing and the phases were seprated by cenl riftlga—
tion at 8 000 r／min for l5 min at a room tempera—
ture．Collected the upper water and repeated the
CI extraction at 4℃ ． Fhen transfered the upper a一
。ueous layer to a new l0 mL tube with a wide—bore
pipette tip．An 2／3 volume of cold isopropanol was
added and mixed properly to precipitate the DNA．
Centrifuge at 10 000 r／min for 10 min．Discarded
the suDernatant，washed the pellet with lml 75
ethanol twice．Dried the pellet and dissolved in 200
肚L TE．The DNA was stored at一2O℃．
1．3 RAPD analysis
Random primers，Taq DNA polymerase，dNTP
were all bought from Sheng Gong Company o{
Shanghai．1 he re action mixl ure consist ed of 1
bufier，2．25 mM MgC12，0．15 mM dN FPs，0．2 M
primer，1．0 U Faq DNA polymerase and 5O ng ge—
nomic DNA per 2O L reaction volume．The ampli—
fication reaction was performed in GeneAmp PCR
svstem 2400(Perkin Elmer Corp．USA)and pro—
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2期 伊艳杰等：利用 RAPD研究桂林桂花品种间的亲缘关系 131
grammed for initial heat denaturation at 94 for 2
min，40 cycles of 94℃ (50 s)，37℃ (1 min)，72℃
(2 min)followed by an extension period of 8 min at
72℃ and then held at 4 ℃ ． A negative control
PCR tube containing all compoents except genomic
DNA was included In all the runs．The amplified
fragments were separated on 1．j agarose gel
with ethidium bromide(O．5 fg／mL)in 1×TAE
buffer at 50 V．The gel was visualized by illumina—
tion with ultraviolet light and photographed．
1．4 Analysis of cultivar relationships
Each ampli fication fragment generated by PCR
was treated as a unit character and scored as pres—
ent(1)or absent(O)．Genetic distances were calcu—
lated between all pairs of entries using Nei’s coef—
ficient of genetic distance(Nei et“ ．，1979)：F一
2Xab／(Xa+Xb)，P一1一F；Where F is the pairwise
similarity coefficient，Xa and Xb are the total Flum—
ber of bands in cultivar A and B respectively，Xab
is the number of ban-is shared by A and B，and P is
the genetic distance between A and B．A dendro—
gram was prepared for the relationships among the
23 cultivars based on the genetic distance matrix by
SAS computer program．
Table l Sequences of 20 random primers
and nutubers of RAPD markers
3000bp
2000bp
1 500bp
1 200bp
1031bp
900bp
800bp
700bp
600bp
500bp
Fig． 1 RAPD patterns amplified by primer$2O25
M DNA marker，100bp DNA ladder；N：Negati~ e control numbers refer to the corresponding nanles of( ／r“ rⅡ cuhivars
2 Results and analysis
2．1 Results of DN A amplification
2O random primers were selected from 100
primers because they could amplificate clear，stable
and repeatable bands．The tota1 of 1 93 distanct ma—
jor RAPD bands，114(59．1 )out of which were
polyrnorphic，were consistently generated from 2O
primers．Between 5 to 1 4 bands were scored perprimer
with an average of 9．6 bands(Table 1)
． Sizes of am～
plified fragments ranged from 400 to 3 000 bp
． Differ—
ent primers had different amplification results
．
Each cultivar gave unique amplification products lo
distinguish it from the other tested genotypes when
several primers were considered．It fully indicated
abundant polymorphism existed between genomes
．
As shown in Fig．1，the polymorphism of 23 fila—
ments was very high and the result was stable after
being repeated for many times．
2．2 Analysis of phylogenetic relationships among
cultivars
Genetic distances derived from pairwise simi～
larity coeficients among the 23 O．fragrans culti—
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132 广 西 植 物 25卷
vars are summarize(t in Table．2．The values of ge—
netic distance ranged from 0．054 8 to 0．576 4，
which illustrated that the genetic difference among
O．fragrayl8 cultivars was very dislinct．The smal一
1est genetic distance was found between O．-厂rⅡ一
grans‘Zi’e’and O．fragrans‘Nongchaoer’，which
had a genetic distance coefficient of 0．054 8．Obvi—
ously，they had close phylogenetic relationship．
The remotest relationship took place between
fragrans‘Nanxi Dangui’and O．fragrans‘Yueyue—
gui’with the genetic distance of 0．576 4．The rela—
tionships between Q fragrans‘Nanxi Dangui’and Q
fragrans‘Sijigui’．0．fragran8‘Guifeihong’and Q
fragrans‘Yueyuegui’．Q fragrans‘Danxin’and
fragrans‘Sijigui’were also remote，whose values of
distances were larger than 0．5．
Table 2 Genetic distances among 23 O。fragrans cultivars
l 2 3 4 ll
1 0．000 0
2 0．078 2 0．O00 0
3 0．109 7 0．1l4 5 0．0O0 0
4 0．1l2 9 0．063 l 0．1l8 l 0．000 0
5 0．228 5 0．2l6 2 u．2l8 3 0．204 7 0．000 0
6 0．216 1 0．227 5 0．220 3 0．198 7 0．067 9 0．000 0
7 0．2O3 7 0．2l9 6 0．2l8 3 0．207 7 O．054 8 0．090 4 0．000 0
8 0．238 2 0．227 8 0．230 6 0．228 l 0．078 4 0．093 7 0．083 2 0．000 0
9 0．237 l 0．229 7 0．220 8 0．219 2 0．092 l 0．096 2 0．106 5 0．09l 7 0．000 0
l0 0．226 5 0．232 7 0．229 l 0．22l 7 0．089 3 0．092 l 0．097 3 0．088 6 0．057 2 0．000 0
ll 0．230 6 0．228 2 0．2l6 l 0．2l8 4 (J．09l 6 0．069 3 0．090 2 0．089 l 0．102 7 0．09l 3 0．000 0
l2 0．226 8 0．223 6 0．2l8 5 0．2l9 7 0．089 0 0．09l 4 0．092 5 0．06l 7 0．098 2 0．092 l 0．089 4 0．000 0
l3 0．204 8 0．2l6 7 0．2O9 9 0．20l 6 0．138 2 0．126 l 0．132 7 0．137 8 0．127 3 0．138 4 0．142 7 0．126 8 0．000 0
l4 0．203 7 0．2l0 8 0．2ll 7 0．187 3 0．132 l 0．130 9 0．13l 4 0．142 3 0．129 l 0．140 2 0．132 5 0．125 7 0．092 9 0．000 0
l5 0．2l0 6 0．2l7 3 0．209 8 0．190 7 0．128 7 0．139 2 0．125 l 0．128 3 0．130 2 0．146 3 0．13l 4 0．158 l 0．098 0 0．097 l
l6 0．2O9 9 0．2ll 3 0．210 7 0．176 5 0．126 3 0．13O 8 0．13l 5 0．132 9 0．140 5 0．145 l 0．132 8 0．149 3 0．O8l 6 0．09l 3
l7 0．207 5 0．208 l 0．2l0 4 0．182 5 ( 15l 7 0．143 4 0．129 3 0．140 7 0．127 l 0．137 8 0．13l 2 0．147 8 0．102 7 0．074 8
l8 0．187 7 0．180 3 0．196 4 0．179 3 (．3l2 8 0．332 7 0．323 l 0．3l8 3 0．330 7 0．328 1 0．3l9 8 0．320 7 0．327 6 0．3l9 7
l9 0．195 l 0．182 4 0．183 l 0．18O 2 O．306 9 0．32l 5 0．327 3 0．3l7 5 0．332 l 0．3l7 3 0．3l8 l 0．326 3 0．32l 5 0．322 5
20 0．189 3 0．187 l 0．1 92 4 0．19O 7 0．3l0 3 0．330 8 0．3l6 7 0．3l7 8 0．326 9 0．3l7 4 0．316 5 0．322 5 0．323 7 0．3l6 8
21 0．181 5 0．180 9 0．179 1 0．152 4 0．300 8 0．318 7 0．317 5 0．309 9 0．324 4 0．309 3 0．318 5 0．319 2 0．313 9 0．318 3
22 0．4l5 3 0．4l8 l 0．420 3 0．432 8 0．272 l 0．324 7 0．373 3 0．3l6 9 0．270 6 0．272 6 0．3l1 9 0．27l 7 0．267 5 0．263 3
23 0．457 l 0．447 3 u．450 2 0．44l 9 0．298 2 0．296 l 0．306 4 0．298 5 0．268 3 0．270 4 0．3l5 3 0．267 4 0．258 l 0．260 2
l5 l 6 l7 l8 l9 2O 2l 22 23
The Single Linkage method cluster analysis
was carried oul by using SAS software based on
the genetic distances of 23 samples．A dendrogram
was developed(Fig．2)． The 23 O．_厂，．ngrn cu1ti—
Vars were diVided int。4 c1usters a 0．1 5 simi1arity
1eve1． Of a1 the 4 groups，3(L“ _厂_OZ Group，
丁 “ ，J rg Group and A rn ￡ nf Group)were
Autumn Division，and the other(Frngr Group)
was Frngrn Division． Their re1ationship groups
based on the RAPD resu1ts were basicallv in accord
on the traditiona1 taxonomy． So RAPD ana1ysis
worked efficiently in th s studv．
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期 伊艳杰等：利用 RAPD研究桂林桂花品种间的亲缘关系
0 0 05 0 i 0 i 5 0 2 0 3
Fig．2 Dendrogram based on RAPD markers
amplified from 23 O．fragrans eultivars
3 Discussions
using the inforescence types and blossoming
habits as the first criterion，and the flower colors as
the second one，Xiang Qi—bai et a1．proposed a sys—
tem in which two divisions and four groups were
recognized，namely，Fragrans Division and Autumn
Division．Fragrans Division is more primitive than
Autumn Division，while the evolutionary sequence
from primitive to advanced in Autumn Division is
Latifolius Group，Thunbergi Group and Auranti—
ncMs Group(Zang et a1．，2002)．Ahhough our con—
clusions in this study held out their viewpoints，
there was difference about cultivar divisions in cul—
tivar group．As it was shown in lhe dengrogram，
cuhivar of O．fragrans‘Taoye Jingui’and O．厂r“一
grans‘Jinlian’was clustered at 0．063 1．The result
showed the closer relationship exited between the
two cuhivars． As far as physiological traits are
concerned，the ovary of O．frngrn1ls‘Taoye Jingui：
is sterile，but 0．fra grans‘J inlian’is fertile．This
trait is regarded as the main basis in classification
of O．fragrans cuhivars． However，according to
our resuIts，the genetic distance between them is
very smal1．Maybe the samples tested in our study
is insufficient，and if we have enough samples，we
will obtain a large number of genotype and offer
more foundation for analysis of genetic relation—
ships among 0．fragrans cuhivars．
RAPD is now widely used in the study of plant
svstematic evolution，phylogenetic relationship and
genetic polymorphism(Lu et a1．，2002)． Although
the stability and reliability of RAPD technique is
suspected，W e think if reaction conditions are opti—
mized and aU reagents are fixed and guaranteed
same in all t he runs，the stable and repeatable re—
suhs will be easily obtained． Our results have
shown that RAPD based classification of O．厂r“一
r“ s cuhivars is an alternative and complementary
approach to the traditional methods for studying
O．fragrans．
References：
Liu YL，Xiang QB．2000．studies on the classification of Os—
“ th“ fragr“ cuhivars[A]． 2O years of science and
technology development of Chinese flowers[C]．Science
Pr ，1：631—642．(in chinese)
Yang KM，Zhu WJ．2000．Osmanthus fragrans Lour．[M]．
Shanghai：Science and Technology Press，8— 12．(in ehi—
nese)
Huang YY，Huang DL． 1949．Theories of flowers[M]．
Shanghai：New centu ry press，(in chinese)
Liu YL．2000．Studies on resources and classification of Os—
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丁 f^ ，27：27—31．(in chinese)
P．Obara—Okeyo& S．Kako．Genetic diversity and identifiea—
ti0n 0f Cvmbidium cultivars as measured by random ampli—
fled D0lymorphic DNA(RAPD)markers[J]．Euphyica，
1998，99：95— 101．
Chen JY，Ning YX，Zhao CH，el a1．1 995．Studies on the
isoperoxidase 0f Osm6z 71th“ fragrans varieties in Hcnan[J]．
Act“H0rt Sin，22(2)：176— 18O．(in chinese)
Zhu C．Liu FY． 1999．To apply RAPD in classification and
differentiation of cuhivar of Osmanthus fragrans Lour．[j]．
Guihaia，19(2)：190—192．(in chinese)
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manth“ fragrt1 713cuhivars[J~．J Huazhong Agr Univ，18
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178 广 西 植 物 25卷
续表 1
种名 习性 及用途 种名 习性 及用途
Species Habit and purpose Species Habit and pu rpose
鸡矢藤 Paederia ca valeriei 缠绕 ，蛘垣 ，常绿 光叶叶子花 Bougainvillea glabra 缠绕 ．廊架，常绿脱花
广西鸡矢藤 P，pertomentosa 缠绕，蝉垣 ，常绿 叶子花 B．spectabilis 缠绕。廊架 ，常绿观花
毛鸡矢藤 P．scandens ev．Tomentosa 缠绕，蛘垣，常绿 羽叶喜林芋 Philodendron 6 p2 n“ “” 气生根，岩坪，常绿
毛钩藤Uncaria hirsute 缠绕，廊架，常绿 深裂喜林芋 P．elgans 气生根．岩蛘．常绿
大叶钩藤 U．macrophylla 缠绕，廊架，常绿 红柄喜林芋P．erubescens 气生根，岩蛘，常绿
钩藤u．r^ 如0 缠绕，廊架，常绿 缎叶喜林芋 P、sloriosum 气生根，岩蛘，常绿
华钩藤U．sinensis 缠绕，廊架，常绿 黑金喜林芋P．martianum 气生根，岩蛘，常绿
马蔸铃 Aristolochia debilis 缠绕，廊架，常绿 攀援喜林芋P．scandens 气生根，岩±羊，常绿
海南马蔸铃 A，hainanensis 缠绕，廊架，常绿 喜林芋P．imbe 气生根，岩蛘，常绿
大叶马蔸铃 A．shakangii 缠绕 ，廊架，常绿 大花清明花 BeauTnonlia grandiflora 缠绕，廊架，常绿观花
大血藤Sargentodoa：a cuneala 缠绕，廊架，常绿 白花鱼藤 Derris alborubra 缠绕．廊架，观叶果
毛叶轮环藤 Cyclea bart,arta 缠绕，蛘垣，常绿 毛鱼藤D．elliptica 缠绕，廊架，观叶果
密花轮环藤 C．densiflora 缠绕，蛘垣，常绿 锈毛鱼藤 D．ferruginea 缠绕，廊架，观叶果
海南轮环藤 C．harnanensis 缠绕 ，蝉垣 ，常绿 亮叶揭阳鱼藤 D．fordii va r，lucida 缠绕 ，廊架 ，观叶果
四JI【轮环藤 C．sulchuenensis 缠绕，蛘垣，常绿 揭阳鱼藤 D．fordii 缠绕，廊架，观叶果
轮环藤 C．racemosa 缠绕，蝉垣，常绿 粉叶鱼藤 D．glauca 缠绕．廊架，观叶果
广西轮环藤 C．sutchuenensis CV．Sessilis 缠绕，蝉垣，常绿 边荚鱼藤 D．marginata 缠绕，廊架，观叶果
金钱吊乌龟 Stephania cepharantha 缠绕，蝉垣，常绿 密锥花鱼藤 D．thyrstflora 缠绕，廊架，观叶果
千斤藤S．hernandifolia 缠绕，蝉垣，常绿 黔桂鱼藤 D．tonkinensis 缠绕，廊架，观叶果
华千斤藤S．sinica 缠绕，烊垣，常绿
3．10使君子 Quiqualis indica L．
属使君子科使君子属落叶蔓性木质藤本。叶对
生，倒卵状椭圆形，先端尖基部圆。穗状花序，悬垂
状，顶生或腋生，夏季开花，初时粉白色后转桃红色，
花期长达 2～3个月。生性强健，蔓性力强，春夏枝
叶浓密，为花廊、拱fJ、围篱或荫棚美化良材。
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