全 文 ：热带亚热带植物学报 2002，10(4)：306—312
Journal of Tropical and Subtropical Botany
红麻及其近缘种的 RAPD分析
郭安平 周 鹏 粟建光
(1．中国热带农业科学院热带作物生物技术国家重点实验室，海南 海1：3 571101
2．中国农业科学院麻类研究所，湖南 长沙 410006)
摘要 ：利用随机扩增多态性 DNA(RAPD)技术分析了木槿属 (Hibiscus)Furcaria组中纤维作物
红麻 (H．cannab／nus)及其 6个近缘种植物的 25份材料。用筛选 出的 16个引物扩增出 192个
RAPD条带，它们表现 出丰富的多态性 。根据得到的 RAPD指纹图谱，计算其 Nei氏相似系数和
遗传距离 ，并构建 了它们的系统树。结果表 明：25份材料可划分为 7个组 ，H．penduriformis和
H．calyphylus 两个种为一组；红麻种分为两个组 ，一组为栽培品种 ，另 1组为野生型材料 ；其余 4
个种各成为 1组。玫瑰麻 (H．sabdarifa)和金线吊芙蓉 (H radiatus)的关系较近，而且两者与红
麻的亲缘关系也较近，而其它四个种与红麻的关系较远。H．trionum与其它六个种的关系较远。
关键词 ：红麻：近缘种；RAPD；亲缘关系
中图分类号：Q94．336 文献标识码 ：A 文章编号 ：1005-3395(2002)04-0306-07
Random Amplified Polymorphic DNA (RAPD)Analyses among
Hibiscus cannabinus and Related Species
GU0 An-p i ng ’ ZHOU Peng SU J i an—guang
(1．National Key Biotechnology Laboratoryfor Tropical Crops，Chinese Academy of Tropical Agriculture Sciences，Haikou
571101，China；2．Institute of Bast Fiber Crops，Chinese Academy of Agricultural Sciences，Changsha410006，China)
Abstract：RAPDs were used to study the relationships among species in Hibiscus sect．Furcaria．
Twenty—five accessions of 7 species were examined． Sixteen primers of arbitrary sequences were
screened from 8O primers for DNA amplification．A total of 1 92 bands were generated,of which
149 bands were polymorphic markers．Dendrogram was constructed using Nei’S genetic similarity
value and MINTS program． The result showed that 25 accessions were clustered in 7 clades． H．
penduriformis and H．calyphylus formed a clade,and 15 accessions of H．cannabinus were
clustered in two clades，one being the cultivars，an d the other，the wild type materials．The rest 4
species form ed 4 individual clades．The most parsimonious dendrogram shows that H．rad／atus is
closely related to H sabdarifa．The closely related species to the cultivars of皿 cannabinus
are H．sabdarifa and H．rad／atus．whereas H．trionum appears to be highly diverged from al
the other taxa．
Received：2001-1 1-13 Accepted：2002-06-26
Corresponding author
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第 4期 郭安平等 ：红麻及其近缘种的 RAPD分析
Key words： Hibiscus cannabinus； Related species； RAPD； Phylogenetic relationship
The genus Hibiscus is a large polymorphic group comprising approximately 400 annual and
perennial species，and they ale classifed into 6 sections(Hochreutiner 1 900)，namely，凡
Alyogne，Abelmcschus，Ketmia，Calyphylliand an d Axanza．Tropical zone an d subtropical zone are
their distribution areast”．
In section Furcaria,there are more than 40 species，most ofthem are tetraploid，some species
are diploid，octoploid or even decaploid，an d their somatic chromosome number ranges from 36
to 188．Among Hibiscus sect．Furcaria species，H．cannabinus(kenaf)and H．sabdarifa(rosele)
are cultivated as fiber cropst”．Kenafis one ofthe main bast fiber crops in China．
Th ere is some valuable gene source in wild species of H． cannabinus， for which the
cultivated species lack，such as some resistance genes(resistance to nematode，insect pest,and
harsh condition，etc．)and superior fiber quality gene“．Because the genome relationships among
the species are not very clear， we lack the efective pathways to tran sferthe valua ble genes into
the cultivars ofH．cannabinus．M orphological characters for most species of sect．Furcaria have
been studied, they were preliminarily classified according to their morphological feature． Th e
chromosome number and ploidy for some species in this section were investigated雎 ． Menzel
and Wilson嘲studied the affmity ofthe species hybridization am ong species of sect． Furcaria,
showing that a few species，such as H．radiatus，H．sabdarifa,were the most closely related to
H cannabinus species．Xiao et al[6]had studied the compatibility of 5 species of Hibiscus。and
selected a interspecific hybrid from the subsequent generations of H．cannabinus x H． tad／at
hybridization, and the chr omosome behavior from Fl to F8 were investigated． Alth ough these
studies have shed some lighton the species relationships am ongHibiscus sect．Furcaria,there is
no an y evidence for the species relationship on DNA molecule level at present． It is evident that
further studies are necessary to as certain the genetic relationships am ong various species．
W illiam s an d Kubelik川 an d W elsh an d McClellandt~ have described simple methods for
assessing genetic variability and for construction of gene maps， based on the am plification of
genetic DNA with single primers of arbitrary nucleotide sequence． Th ese primers have been
shown to detect polymorphisms in the absence of specific nucleotide sequence information in
DNA．In RAPD (random am plifcation of polymorphic DNA)，fingerprinting technique，the
oligonucleotides are randomly produced and do not require any DNA target information． Th e
observed fingerprints for a given DNA sample will depend on the len gth an d sequence of the
primer，as well as on the optimization ofreaction conditions．
In the past a few years， many an alysis have been perform ed using RAPD to solve genetic
relationships am ong diferent species,such as Brassica~ q，riceB~,A vena sterilis 2I mahoganies
,waterm elon 04]， Pisum 啕， etc． Th e polymorphisms can provide unique information for
1)imcxnntional JuteOrganization．ProceedingsoftheWorkshoponApplicationofBioteclmologyintheImprovementofJu~
KemfandAlliedFibers-PhaseLBeijing，China，23-25Novem bel；1998．45-46．
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308 热带 亚热 带植物 学报 第 10卷
classification．The aim of this work is to study genetic relationships among H．cannabinus and the
6 related species ofHibiscus sect．Furcaria using RAPD．
1 M aterials and methods
Plant materials Seven species including 25 accessions in Hibiscus sect．Furcaria were
used．Al the materials were obtained from International Jute Organization(IJO)and Institute of
Bast Fiber Crops，Chinese Academy of Agricultural Sciences(IBFC，CAAS)．A complete list of
species，accession number and their chromosome number is presented in Table 1．
DNA isolation and characterization DNA was prepared from fresh tender leaf
tissues，and the method described by Pich and Schubesn was adopted for DNA isolation．Using
a Backrnen DU一7 spectrophotometer to measure the value of OD26o and OD280 for DNA
samples， the concentration and quality was tested． Using the 入DNA／Hind 1II as molecular
standard， the size of DNA molecular was evaluated by electrophoresis on 0．7％ agarose ge1．
Samples were diluted in TE(1 0 mmol／L Tris—HC1，pH 8．0，1 mmol／L EDTA)to achieve a final
concentration of40 ng la 1。。．
Table 1 List of species studied，their accession number，chromosome number and source I
Species Number Accession name Chromosome number~ I Source
- “ abinv~s 1 J
． 1．113 36 America
2 Qingpi No．3 36 Vietnam
3 722 36 China i
4 85．98 36 China I
5 Alian 36 0atar l
6 74．1 36 China l
7 PI248901 36 America 1
8 PI248895 36 America l
9 KB6 36 China
10 84201 36 China
11 KB5 36 China
12 85．239 36 Kenya
13 85．335 36 Kenya
14 85-133 36 Kenya
15 85．225 36 Kenya
． sabdarifa 16 85．122 72 China
17 H152 72 Thailand
18 H153 72 Thailand
19 H181 72 Thailand
H．radi~us 20 H158 72 Kenya
H．trionum 2 1 H003 72 Kenya
H．acetosella 22 H071 72 Kenya
． penduriformis 23 H321 72 Kenya
24 H322 72 Kenya
． c 、ph vllv~s 25 H334 72 Kenya
嚣
l l - 。 粒 §瓣涸■■鳃 涵 萋
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第 4期 郭安平等：红麻及其近缘种 的 RAPD分析 309
RAPD analysis RAPD analysis were performed using 16 primers obtained from
Operon Technologies Corporation． These 1 6 primers were screened from 80 primers including
OPE， OPG， OPH and OPN four series by
using two DNA samples for am plification
reac tion． The nueleotide sequence of each
ran dom primer is shown in Table 2．
Polymerase chain reactions(PCR)were
perform ed in 25lal volumes containing
10 mmol／L Tris．HCl， 1％ Triton X．100，
1．5 mmol／L MgC12, 0．1 mmol／L each of
dATP，dCTP，dGTP an d dTTP，40 ng DNA
sam ple， 1．5 units of Taq polymerase an d
5 pmol／L primer． A negative control
containing water instead of template DNA
was included in each reac tion set． Th e
am plificatiOIlS were carried out in a PERK
DNA Th erm al Cycler an d programmed for
5minat95cI=predenature． 5 cyclesof1 min
at 94cI=to denature． 1 min at 36cI=for
Table 2 List ot prim ers and their respec~ve
oligonudeotide sequence
annealingprimer,and2min at72cI=forextension,then40 cyclesof30 seconds at94cI=．1 min at
36cI=，2 min(10 min for the last cycle)at 72℃，after the 45 cycles were completed,10 u l ofeach
DNA am plification products was an alyzed by electrophoresis on 1．4％ agarose gel containing
ethidium bromide(0．5 ug ml- )in 1×TAE bufer at 5 V cm。and recorded by UV photography~阍．
n molecular standard us ed Was the PCR markers obtained from Sino-American
Biotechnologies Corporation(SABC)．
Data analyses Only clear am plified DNA ban ds were scored． Th e occurrence of a
specific ban d ofamplified DNA Was scored as 1 an d absence aS 0 within a fingerprint．Th erefore．
a sequen ce of0’S an d IS Was generated for each primer／ac cession to form a data matrix．Th e da ta
f0r al the primers were used to estimate the Nei’S simi larity based on the number ofbands． A
dendrogram based on similarity coefi cients was generated by using the parsimony．
2 Results and discussion
2．1 Polymorphic bands
RAPD polymorphisms were evaluated using 16 primers for each species／ac cessions． Three
replications were perform ed for each primer． Th ere were 192 ban ds for 16 primers in tota1．
Among all the ban ds，149 markers were polymorphic while the other 43 markers were
monomorphic．Th epolymorphicban ds comprised 77．6％ ofthetotalban ds．Severalpolymorphic
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31O 热 带 亚热 带植 物 学报 第 1O卷
bands were observed for each primer．M ost primers produced multibanded fingerprints．Figures 1
and 2 illustrated the results obtained for primer OPG·07 and OPN-1 5， respectively． The data of
these bands were used in similarity evaluation． The similarity matrix was obtained after
multivariate analysis using Nei’S coefficient, then these similarity coeficients were used to
produce dendrogram by parsimony．
1543
994
695
515
377
237
M 1 2 3 4 5 6 7 8 9 10 l1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 _
1543
994
695
515
377
237
一
Fig．1 Fingerprinting analysis for 25 accessions of Hibiscus sect．Furcaria using primer OPG一07
Lables for each lan e were the accession numbers listed in Table 1．M：PCR markers
2 3 4 5 6 8 14 15 16 17 22 23 24 25 一
Fig．2 Fingerprintinganalysisfor25 accessionsofHibiscus sect．FurcariausingprimerOPN一15
Lables for each lan e were the accession numbers listed in Table 1．M：PCR markers
2．2 Genetic distance analysis
Tlle most parsimonious dendrogram obtained for all primers combined is presented in Figure
3．From the dendrogram，25 accessions are clustered in seven clades，namely，A，B，C，D，E，F，
and G．Clade A iS the accessions of日．sabdarifa．In this clade．four accessions Can be divided
into two subgroups at similarity of O．93． and the first subgroup is three wild type accessions
(H153．H181 and H152)，the second subgroup iS the cultivar ofrosele 85-122．CIade B iS the
species日．rad／atus．Fifteen accessions of日．cannabinus are clustered in two clades，one(C)iS
the II cultivars，the other(D)is the wild type accessions．It reveals that among these species，the
cultivars are diferent from the wild materials in genome， in fact,the variation of morphological
characteristics iS great betw een the tw o types of materials． Clade E iS 日．acetoseUa． Clade F
included tw o species，which Can also be divided into tw o subgroups at the similarity of0．87，one
is the accessions of species H．pendurimormis，an d the other，species H．cdyphyllus，which
indicate that there is close genetic relationships between the two species．日．trionum(Clade G、
appearstobethemostdistant speciesfrom the othertaxa．
H．radiatus and H．sabdarifa were the most closely related species with the cultivars ofH．
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第 4期 郭安平等：红麻及其近缘种 的 RAPD分析 3l1
cannabinus． The result is in agreemem with the conclusion got from species hybridization by
M enzel et a1．团，and is also in agreement with the chromosome behavior observed in F1一Fs from
cannabinus× radia~ 嘲．
The value ofgenetic distance between H．radiatus and H．sabdarifa is smal，and these two
species are also quite sim ilar in some morphological characters【5】．
The accessions within the same species， such as H．sabdarifa，H．cannabinus and H．
penduriformis，appear to be closely afiliated, which is good evidence for the caliber of
experimental data．
80 85 90 95 100 Simi Iarity (’1)
Hl53
Hl8l
Hl52
85．122
Hl58
P1248901
Pl248895
J．I．1l3
85．98
———-_1
H．sabdarifa
QingpiNa3
KB6 H．cannabinus
722 (CuIt；avar)
74．1
Alia．
84201 l
KB5 ．-—-—--·——一
85．335 ————__1
85．1 33 H．cannabinus
85．239 (Wi Id type)
85．225 ．．．．．．_-J
ti07 l H．acetosella
H321 H．penduriformis
H322 H．pen&triformis
11334 H．calyphyllus
H003 H．triomtm
Fig．3 Themostparsimoniousdendrogram showingthe relationships among25 accessionsofHibiscus sect．Furcar／a
]
f
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312 热 带亚热带植 物学报 第 10卷
In this study， the power of RAPDs to reveal polymorphisms among 7 species of Hibiscus
sect．Furcar／a is clearly revealed．W e attempt to use RAPD markers as a tool to implement studies
of molecular systematics in Hibiscus． M ore accessions from Hibiscus species and more species
which are not Considered in present experiment will be included in our future studies，and the use
of more primers will provide greater resolution of the relationships of these taxa．
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