全 文 ：热带亚热带植物学报 2005，l3(3)：246—252
Journal ofTropical and Subtropical Botany
用RAPD和ISSR检测的橡胶树野生
种质和栽培品种的遗传多样性
安泽伟，孙爱花，程 汉，黄华孙 ，方家林
(叶1闵热带农业科学院橡胶研究所，国家橡胶树育种叶1心，农业部热带作物栽培生理学重点实验室，海南 儋州 571737)
摘要：用 l9个 RAPD引物和 12个ISSR引物对 14份野牛橡胶树种质和我国的37份栽培品种进行了遗传多样性分
析。RAPD引物 产牛 132条带，多态性带占88．6％，卡H似系数变化范围在O．432—0．947。ISSR引物 产牛 101条带，多
态性带占87．1％，相似系数为O．505—0．941。平均基因杂合度分析表明野牛种质比栽培品种具有较高的遗传多样性。根
据UPGMA法对 5l份材料进行聚类分析，结果表明，ISSR分析中所有材料可分为2类：第 ‘类为野牛种质，第 lI类为
栽培品种：而 RAPD分析中野牛种质和栽培品种 能被分为明显的两人类。虽然ISSR和RAPD的聚类分析结果存在
蓐异，但对两种方法进行的卡H关分析表明，他们之间仍存在极显著卡H关性，相关系数为O．574。品种PR107、热研2l7等
·些栽培品种可以通过特异带在5l份供试材料中被区分开。这些结果可以对橡胶树的育种上作起到 ‘定的指导作
用，同时RAPD和ISSR技术也是进行橡胶树品种鉴定和遗传多样性研究的有效手段。
关键词：橡胶树：种质资源：遗传多样性：RAPD：ISSR
中图分类号：Ol6 文献标识码：A 文章编号：1005—3395(2005)03—0246—07
Genetic Diversity among W ild and Cultivated Accessions of
Hevea brasiliensis(Rubber Tree)Detected by RAPDs and ISSRs
AN Ze—wei，SUN Ai—hua，CHENG Han，HUANG Hua—sun ，FANG Jia-1 in
(Rubber Research Institute，Chinese Academy of Tropical Agricultural Sciences；State Centerfor Rubber Breeding；
Key Laboratory of Cultivation Physiologyfor Tropical Crops，Ministry ofAgricultural，Danzhou 571737，China)
Abstract： In order to select suitable Heyea accessions for extending genetic base of Chinese rubber tree in future
breeding schemes．the genetic diversity Of l 4 wild accessions and 37 cultivated clones were detected by RAPD and
ISSR．Thirty—one reliable primers(1 9 RAPD primers and 1 2 ISSR primers)were chosen．Based on RA PD，a total
Of l 32 bands were generated， polymorphic bands accounted for 88．6％， and similarity coeficient ranged from
O．432 to 0．947 among all accessions．Otherwise based on ISSR，1 0 1 bands were produced。polymorphic bands
accounted for 87．1％．and similarity coe瓶cient was 0．505 to 0．941．The wild accessions showed higher polymor—
phism than cultivated clones according to the average heterozygosity．Based on ISSR，the 5 1 accessions were
divided into 2 clusters according to unweighted pair—group method with arithmetic averages(UPGMA)cluster
analysis：Cluster I contained 9 wild accessions and Cluster II consisted of 37 cultivated clones and 5 wild
accessions．But all of the accessions can not be divided into wild cluster and cultivated cluster according to RA PD．
Some cultivated clones，i．e．PR 1 07，Re yan 2 l 7，could be screened from 5 1 accessions．The significantly high
correlation between RAPDS and ISSRs among 5 l accessions was observed (r：0．574)，although the diferences
betw een the RA PD and ISSR dendrograms were observed． It iS proven that all of the results can be used in Heyea
breeding programs，and RA PD and ISSR can be used in clona¨ dentification and diversity study of Hevea．
Received：2004——09—-23 Accepted：2005——0 1——3 1
Foundationitem：SupportedbyNationalScienceBasicResearchandSocialCommonwealResearchFoundation(2003DEB6J075，2003DIB7J066)
Coresponding author
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第3期 安泽伟等：用RAPD和ISSR检测的橡胶树野生种质和栽培品种的遗传多样性 247
Key words：Hevea brasiliensis；Germplasm；Genetic diversity；RAPD；ISSR
Rubber tree(Hevea brasiliensis Muel1．Arg．)，the
main source of natural rubber，is indigenous to the
Amazon basin of South American． The English man，
Wickham H． K． collected a lot ofHevea seeds from
Amazon basin in 1 876．He grew the Hevea seedlings
in England， and transplanted them in Indonesia，
Malaysia，Singapore and Sri Lanka．Today rubber tree
is extensively cultivated in Southeast Asia， in which
the yield of nature rubber is more than 90％ of the
total al over the world．The cultivated clones(known
as Wickham clones) of Southeast Asia were al
derived from 22 seedlings surviving from the
Wickham collection in 1 876， and showed a narrow
genetic base【l】．Therefore，the International Rubber
Research and Development Board(IRRDB)organized
a survey of Hevea wild germplasm in the Amazon
forest in 198 1．Exploitation of these genetic resources
requires the knowledge of their genetic diversity．Now
molecular markers have been used to detect the
genetic variation of rubber tree，because they are more
precise，eficient than morphological markers．
Genetic diversity of H． brasiliensis has been
detected by several molecular markers，i．e．，isozyme E1，
I LP ．minisatellite E ，SSR『61．AFLP[7_and RAPD[81．
Inter—simple sequence repeats(1SSR)have not been
reported in Hevea genetic study yet， but it has been
used in other crops，e．g．rice[9]，Euzalyptus『l01，sweet
potato~”1，potatoD2I，sesame 1
In China，the cultivated rubber clones have a very
narrow genetic base，most of which were derived from
several foreign clones，such as RRIM600，PR107．The
narrow genetic base has hold back the development of
Chinese rubber breeding． In order to extend the
genetic base of Chinese rubber clones，more and more
wild rubber germplasm should be exploited in
breeding． In this study， we evaluated the Hevea
diversity by RAPD and ISSR among 1 4 wild germ—
plasm(1 98 1 Amazonian accessions)and 3 7 cultivated
clones．The objective of this study was to give more
inform ation to select suitable parents for extending
rubber genetic base in the future breeding schemes of
China
1 Materials and Methods
Plant materials This investigation was
based on 14 wild accessions and 37 cultivated clones
(Table 1)．The 14 wild accessions come from the 1981
Am azonian collections，which have some outstanding
traits e．g．fast—growing，cold resistance．The 37 culti—
vated clones are elite rubber clones in China． A11 of
the 5 l accessions have been conserved in state germ —
plasm garden in Rubber Research Institute， Chinese
Academy of Tropical Agricultural Sciences．
DNA extraction Total genomic DNA were
isolated from fresh young leaves using modified
CTAB extraction bufert8]．
RAPD Nineteen primers were selected in
this analysis． PCR was carried out in a 1 0 ixl volume
containing 50 mmol／L KC1，10 mmol／L Tris—HC1，
0．1％ Triton X·100，2 mmol／L MgC12，0．1 mmol／L
dNTPs，O．34 ixmoVL primer，0．5 U Taq Polymerase，
template DNA 30 ng，ddH20 6．5 ix1．The reaction was
run for 45 cycles(denaturing at 94~C for 4 min，10
cycles each consisting of denaturing at 94。C for 30 s，
annealing at 36℃ for 30 s，extension at 72℃ for 70 s；
35 cycles each consisting of denaturing at 94~C for
20 s，annealing at 36℃ for 20 s，extension at 72。C for
60 s；a final extension at 72~C for 7 min)．The ampli—
fication products were separated on 1．5％ agarose gels．
stained with ethidium bromide，visualised with ultra—
violet light and photographed．
ISSR Twelve primers were selected in this
analysis． PCR was carried out in a 1 0 ixl volume
containing 50 mmol／L KC1，1 0 mmol／L Tris·HC1，
0．1％ Triton X一100，2 mmol／L MgC12，0．1 mmol／L
dNTPs， 0．4 ixmol／L primer，0．5 U Taq Polymerase，
template DNA 30 ng，ddH2O 6．64 ix1． The reaction
was run for 45 cycles(an initial denaturing at 94~C for
4 min，35 cycles each consisting of denaturing at 94~C
for 30 s，an ealing at 50℃ for 30 s，extension at 72℃
for 70 s； a single extension at 72℃ for 7 min1．The
amplification products were separated on 2％ agarose
gels， stained with ethidium bromide，visualised with
ultraviolet light and photographed．
Data analysis Fragments amplified by
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248 热带亚热带植物学报 第 l3卷
Table 1 The origins of wild accessions and cultivated dones of brasU／ens／s used in this study
RAPD and ISSR primers were scored as present(1)
and absent(0)．Genetic similarity(GS)were estimated
according to Simple Matching Coeficient based on
the probability that the amplified fragment from one
genotype will be present in another genotype， and the
fragm ent can’t be amplified in both genotypes． GS
( d)／( 6+c+d)，(0：number of shared fragm ents，6：
num ber offragm ents in line A，c：num ber offragm ents
in line B， and d：num ber ofabsent fragm ents in both
line A and line B、． The average heterozygosity was
evaluated according to Nei M．【 ．Correlation between
assays was calculated using the SPSS software
package． Cluster analysis was performed with the
NTSYS—pc software package based on UPGMA
(unweighted pair—group method with arithmetic
average)．
2 Results
2．1 Level of polymorphism
In this study, 5 1 accessions were detected by
means Of l9 RAPD primers and 12 ISSR primers． A
total of l32 bands were generated 、Ⅳith l9 RAPD
primers(Fig．1)，and 7 bands were detected per primer．
Ofthem，l 17 bands(88．6％)were polymorphic．10l
bands were scored for ISSRs(Fig．2)，and 87．1％of
those were polymorphic． On average， 8 bands were
detected per ISSR primer．The number ofpolymorphic
bands based on RAPD was 95(79．2％、 in wild
accessions，and it was 92(80．0％) in cultivated
accessions． The num ber of polymorphic bands based
on ISSR was 84(84％)in wild accessions，and it was
64(68．8％)in cultivated accessions(Table 2)．
2．2 Genetie relationship of 51月 vea accessions
Similarity coeficient was calculated based on
polymorphic data． Based on ISSR， the coeficient
ranged from 0．505 to 0．94 l among all accessions．and
it was 0．432 to 0．947 according to RAPD (data not
showed)．Wild accessions showed lower similarity and
hi er polymorphism than cultivated clones．Based on
ISSR． the coef6cient ranged from 0．505 to 0．87 l in
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筑3圳 叠坪仆等：川 RAPDt：rtISSR椅删l0悸胶拊野 计埙和抵培，j 『lf){J趟fE引干性
Fig 2 ISSR pattern_IralI 5l acc [m‘bv plimet 83fi
Marker is 200 bp ladder m~uker
wild accessions．and 1t ranged from ()．663 to 0 94 1 ii1
cullivaled clones．Based on RAPD．the eoe娟 cient was
0．523 to 0 87 I in wild accessions．and lt was 0．530 to
0 947 in cultivated clones． The dendrogram revealed
two distinct clusters accol d[ng to ISSR fFig 3)：Cluster
I consisted of 9 wild acecssiols； (?luster II comprised
of 37 cultivated clones and 5 wild accessions．Other-
wise the 5 l accessions ca『-’t be divided ihie wild
cluster and cultirated cluster according to RAPD(Fig
4)
2．3 Comparison between a~ays
To compare the resulls obtained wilh the two
techniques，we tOSted Pearson correlations The result
showed that 57 4％ of the pairs of genot}?es were
ranked in the sanle order between RAPD and ISSR
3 Discussion
The 5 l accessions were distinctly separated into
two clusters according to ISSR：cultivated clone c]tlster
and wild accession cluster．This result js consistent
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250 热带亚热带植物学报 第 13卷
TabIe 2 Number of total and polymorphic bands generated by RAPD and ISSR among 51 accessions
Fig．3 Dendrogram of 5 1 Hevea accessions based on ISSR markers
with the pedigree information of 5 1 accessions． W ild
accessions，i．e．MT／IT／18 31／125，MT／IT／16 34／15，
AC／S10 37／93，AC／X／21 64／177，RO／C／8 24／272，are
closely related to cultivated clones， and similar result
has been detected by RFLP【l 51．Based on RAPD．the 5 1
accessions can’t be clearly separated into cultivated
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第3期 安泽伟等：用RAPD和ISSR检测的橡胶树野生种质和栽培品种的遗传多样性 25l
哪 0 o|2
Fig．4 Dendrogram of 5 1 Hevea accessions based on RAPD markers
cluster and wild cluster．The reason may well be that
the two markers are generated by different target
fragments．Increasing the large number ofpolymorphic
primers to detect all accessions might give beter
solutions．
The polymorphic data unambiguously showed
that cultivated clones had less polymorphism than
wild accessions．Based on RAPD，the average hetero—
zygosity was 0．406 in wild accessions．and was 0．3 83 in
cultivated accessions． It was 0．425 in wild accessions
according to ISSR．but was 0．400 in cultivated acces—
sions． The similar results have already been reported
by Bease P．et a1．【 and Lekawipat N．et a1．116】．Despite
their narrow genetic base and high level of inbreeding，
some cultivated clones could be screened from 5 1
accessions，i．e．PR 1 07，Re yan 2 1 7．This result indicated
mat RAPD and ISSR markers could be used to
identify cultivated clones． However， to increase the
precision of cultivated clones identification， much
more polymorphic primers should be needed．
Knowledge of genetic variation and the genetic
relationship between genotypes is an important consi—
deration for eficient utilization of germplasm
resources．Furthermore，it is important for the optimal
design of plant breeding programs， influencing the
choice of genotypes to cross for the development of
new populations．The genetic analysis of 5 1 acces—
sions revealed that wild accessions had more
variability than cultivated clones． Since all of the 5 1
accessions belong to H．brasiliensis，we should select
the parents， which have distant genetic relationship，
from cluster I and cluster II for cross breeding． Thus
we can exploit the wild germ plasm to broaden the
genetic base of cultivated rubber tree． Genus Hevea
has nine species． All of them， H． brasiliensis can
produce the most nature rubber．The other species has
some prospective traits，for example，H．benhamiana
Can resist Microcyclus ulei and Phytophthora
palmivora．In future breeding schemes，we can exploit
not only H．brasiliensis but also other useful species．
Molecular—based estimates of GS will allow plant
breeders to make inform ed decisions regarding the
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252 热带亚热带植物学报 第 13卷
choice of genotype to cross， but we must choose a
suitable assay．According to Vos et a1．【l7J，the ideal
fingerprinting assay should require no prior sequence
knowledge of research objects．While RAPD and ISSR
primers are random primers， and meet these
requirements．In this study，we got the reliable results
by RAPD and ISSR， and the significantly high
correlation between RAPDs and ISSRs among 5 1
accessions was observed r=0．574)． although
differences were observed between the RAPD and
ISSR dendrograms． It was proven that RAPD and
ISSR markers could be used in Hevea clonal
identification and diversity study．
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