全 文 ：　遗 传 学 报　Acta Genetica Sinica , January 2003 , 30 (1):81～ 87 ISSN 0379-4172
收稿日期:2002-08-05;修回日期:2002-10-08
基金项目:国家自然科学基金资助(No.39970081)[ Supported by the National Natural Science Foundation of China(No.39970081)]
作者简介:庞晓明(1973-),湖南宁乡人 ,博士 ,专业方向:生物技术。 E-mail:xmpangcn@gahoo.com
①　通讯作者。 E-mail:dxxwwlj@public.wh.hb.cn
Phylogenetic Relationships Among Citrus and Its Relatives
as Revealed by SSR Markers
PANG Xiao-Ming , HU Chun-Gen , DENG Xiu-Xin①
(National Key Laboratory of Crop Genetic Improvement , Huazhong Agricultural University ,
National Center of Crop Moleculor Breeding , Wuhan　430070 , China)
Abstract:Phylogenetic relationships among 29 accessions belonging to Citrus , Poncirus , Fortunella , Microcitrus , Eremocitrus , At-
lantia and Severinia were investigated using SSR markers.Seven SSR primers generated 114 polymorphic alleles , with an average of
16.3 alleles per primer.Cluster analysis via neighbour-joining method showed that Microcitrus was close to Citrus;Poncirus was
distant from Citrus ,which suggested that Poncirus could not be derived from Citrus.High frequency of the homozygous SSR locus
supported the species status of Fumin trifoliate orange.Seperation of neither Papeda and Citrus nor Archicitrus and Metacitrus was
well resolved.The present work confirmed citron , pummelo and mandarin as basic species of cultivated citrus since they could be
placed into three distinct clusters.
Key words:citrus;fumin trifoliate orange;microcitrus;SSR;phylogenetic relationship
用 SSR标记研究柑橘属及其近缘属植物的亲缘关系
庞晓明 , 胡春根 , 邓秀新①
(华中农业大学作物遗传改良国家重点实验室 , 国家农作物分子育种中心 , 武汉　430070)
摘　要:用 SSR标记分析了 29份柑橘属及近缘属植物的亲缘关系。7 对 SSR引物在 29 个样品中扩增得到 114 个
等位基因 ,平均每个位点有 16.3 个等位基因。计算匹配系数后用邻接法进行聚类 , 结果表明 ,澳洲指橘与柑橘属的
亲缘关系很近;SSR位点的高纯合频率支持富民枳种的地位;枳与柑橘属的关系较远 , 枳不大可能是从柑橘属衍生
而来;Swingle 的亚属的划分以及田中的原生柑橘类和后生柑橘类的划分界线都不清晰;现代栽培柑橘的起源与大
翼橙关系密切;柑橘属的枸橼 、柚和宽皮橘都能很好地分离 , 支持其为现代栽培柑橘的 3个基本种的观点 。
关键词:柑橘属;富民枳;澳洲指橘属;SSR;亲缘关系
中图分类号:Q949　　　文献标识码:A　　　文章编号:0379-4172(2003)01-0081-07
　　Citrus is one of the major fruit crops in the world.
However ,Citrus taxonomy and phylogeny are very compli-
cated , conflicting and confusing ,mainly due to apomixis ,
widely sexual compatibility between Citrus and related
genera , the high frequency of bud mutations and the long
history of cultivation and wide dispersion[ 1] .Before mid
1970s ,Citrus taxonomy was mainly based on morphologi-
cal and geographical data , resulting in various taxonomic
system , among which those of Swingle[ 2] and Tanaka[ 3] are
most widely accepted.In the mid 1970s , a comprehensive
numerical classification study based on 146 morphological
and biochemical characteristics[ 4] and studies of Scora[ 5]
proposed that cultivated Citrus comprises only three basic
species , i.e., citron(C.medica),mandarin (C.reticula-
ta)and pummelo (C.grandis;now C.maxima), later
C.halimii added as the fourth basic species[ 1] .The other
biotypes were derived from hybridization among these true
species or between them and species of the subgenus
Papeda or closely related genera.The other species names
are retained as “convenient species”[ 1] .
In recent several decades , plant molecular systemat-
ics developed rapidly , which has been employed in the
phylogenetic relationship studies of Citrus.The application
of RFLP , ISSR ,SCAR and PCR-RFLP of specific region
of organelle genome in Citrus phylogenetic relationship
have brought new prospect to it[ 6] .However , the results
are not always in agreement.Combining analysis with sev-
eral approaches will probably better improve the accuracy
of phylogenetic inference.Of the many molecular tech-
niques available to researchers , simple sequence repeats
(SSRs) or microsatellites is becoming increasingly
widespread because it is codominant ,highly polymorphic ,
frequently and evenly distributed throughout the genome.
And it was regarded to be the most reliable marker.It has
been used in the phylogeny studies of many plants such as
Glycine[ 7] ,potato[ 8] and Vitaceae[ 9] .But there is no re-
port about the application of SSR markers in phylogenetic
relationships of Citrus and its relatives as yet.China is one
of the most important origin places of Citrus with abundant
germplasms.Herein SSR markers were utilized to analyze
29 accessions of Citrus and six related genera , aiming to
provide new data for the study of phylogenetic relation-
ships of Citrus.
1　Materials and Methods
1.1　Plant materials
19 accessions of Citrus and 10 accessions from six
related genus were included(Table 1).The plant materi-
als were collected from five places and DNA of three ac-
cessions were donated by Dr.DQ Fang (Table 1).
1.2　DNA extraction
About 3 g of fresh young leaf tissue were collected
from each plant ,which were kept at -76℃until use for
DNA preparation.The leaf was grounded to fine powder
with the aid of frozen in liquid nitrogen.A modified SDS
method described by Chen et al.[ 10] was used for DNA
extraction.DNA concentration was determined with the
absorbance in 260 nm using spectrophotometer UV 1601
(Shimadzu Inc., Japan).The DNA templates were diluted
to 25 ng/μl.
Table 1　Plant material used in the study
Code Common name Swingle system Sourceaa)
1 At lantia Atlantia monophylla DC. A
2 Australia f inger lime Microcitrus australasica A
3 Australia dessert lime Eremocitrus glauca Swingle A
4 Chinese box orange Severinia buxifolia Tenore A
5 Zaoshi trifoliate orange P.trifoliata Raf. C
6 Fumin P.polyandra Ding et al. D
7 Flying dragon P.trifoliata Raf. B
8 Tanghe trifoliate orange P.trifoliata Raf. D
9 Meiwa Kumquat Fortunella crassifolia B
10 Kahsi papeda C.latipes Swingle F
11 Maritius papeda C.hystrix DC E
12 Honghe papeda C.honghensis Y L D L A
13 Ichang papeda C.ichangensis Swingle A
14 Lime C.aurantifolia Swingle B
15 Valancia sweet orange C.sinensis Osbeck B
16 Sour orange C.aurantium Linn. B
17 Rough lemon C.jambhiri Osbeck B
18 Shatian pummelo C.maxima(Burm.)Merril A
19 Grapefruit C.paradisi Macf. B
20 Lime C.aurantifolia Swingle B
21 Daoxian wild tangerin C.reticulata Blanco A
22 Bendizao tangerin C.reticulata Blanco B
23 Changsha tangerin C.reticulata Blanco F
24 Tachibana C.tachibana Tan. B
25 unnamed C.halimii Stone B
26 Small citron C.medica Linn. A
27 Hongkong kumquat F.hindsii B
28 Lemon C.limon Burm. B
29 Red lemon C.limonia Osbeck A
　a)A:Citrus Research Institute , Chinese Academy of Agricultural Science
(Chongqing ,China);B:Citrus Research Institute ,Huazhong Agricultural Uni-
versity(Wuhan , China);C:Yichang , Hubei , China;D:Tanghe , Henan , Chi-
na;E:Kunming , Yunnan , China;F:Donated by Dr.Fang from UCR , River-
side , USA
1.3　SSR
1.3.1　PCR amplification
A total of seven primer pairs were used(Table 2).
The primer pairs were synthesized by Sangon Inc.(Shang-
hai ,China)according to published sequences of Kijas et
al[ 11] .The PCR amplifications were performed in 20 μl
reaction volume containing 10 mmol/L Tris-HCl (pH
82 遗　传　学　报 30 卷　
8.3),50 mmol/L KCl ,1.5 mmol/L MgCl2 ,1.0 unit Taq
polymerase (Sangon Inc.), 0.2 mmol/L of each dNTP ,
0.1 μmol/L of each forward and reverse primer ,50 ng of
template DNA and sterilized double stilled water.PCR
amplifications were performed in a PTC 200 thermocycler
(MJ Research Inc.,USA)with the following cycling pa-
rameters:an initial denaturation step at 94℃ for 5 min ,
followed by 30 cycles at 94℃ for 1 min , an annealing
temperature at 55℃(TAA33 45℃)for 1 min and an ex-
tension at 72℃ for 1 min ,with a final extension step at
72℃ for 5 min prior to being holded at 4℃.
Table 2　Polymorphism of the SSR primers
Primer Alleles number PIC
TAA1 10 0.521
TAA15 24 0.750
TAA52 17 0.864
AGG 17 0.613
TAA33 9 0.473
TAA27 11 0.458
TAA45 26 0.858
total 114 　　　　NA
mean 16.3 0.648
1.3.2　Detection of the SSR products
The PCR products were first run on a 1.5% agarose
gel and visualized with ethidium bromide in order to de-
tect amplification success.If amplification was successful ,
the PCR product was run on a 0.04 mm thick 6% poly-
acrylamide denaturing gel.Gels were pre -run at 60 W
for 30 min.The samples were loaded and run under the
same conditions for about 1.5 h.Fragments were visual-
ized by the silver-staining method according to the man-
ual of Silver Staining Kit Q4132(Promega ,USA).
1.3.3　Statistical analysis
SSR was scored as A for presance and T for absence
manually.Diversity level of locus were evaluated with the
polymorphic information content(PIC=1-∑n
j=1
P
2
i)[ 12] .
The genetic distance was calculated as p-distance of nu-
cleotide acid by MEGA2
[ 13] , equivalent to that estimated
with a simple matching coefficient , the proportion of
shared A s and T s subtracted from 1.Thus a matrix was
constructed ,which are available upon request.Neighbor-
joining method was used to cluster and bootstrap[ 14] esti-
mates were calculated for 500 re-samplings.
2　Results
2.1　Polymorphism of the SSR markers
Seven pairs of SSR primers generated 114 alleles , all
of them were polymorphic.The SSR profile generated by
primer TAA1 was shown in Fig.1.Primer TAA33 ampli-
fied nine alleles , whereas primer TAA45 amplified as
many as 26 alleles.An average of w 16.3 alleles were am-
plified per primer.Primer TAA52 had the highest PIC
(0.864), while TAA27 had the lowest (0.458),with a
mean PIC of 0.648(Table 2).
Fig.1　SSR profile of 29 Citrus accessions using primer pair TAA1
Number refers to the samples listed in Table 1;M:100 bp DNA ladder(promega)
83　1 期 庞晓明等:用 SSR标记研究柑橘属及其近缘属植物的亲缘关系
2.2 　Phylogenetic relationships revealed by SSR
markers clustering
　　The highest genetic distance (0.219)was observed
between C.ichangensis and Flyingdragon trifoliate or-
ange ,while the lowest (0)was observed between Zaoshi
trifoliate orange and Tanghe trifoliate orange.Based on
pairwise diatances , a dendrogram was constructed using
neighbour-joining method ,which is presented and only
bootstrap values higher than 40% were added on the
nodes(Fig.2).Neither the separation of subgenus Pape-
da and Citrus nor that of subgenus Archicitrus and Metac-
itrus by Tanaka were clearly resolved in the dendrogram.
C.reticulata , C.maxima and C.medica were separated
into three distinct clusters , supporting the three basic
species concept
[ 4] .It could be seen from Fig.2 that all
the accessions could be grouped into ten clusters:(1)At-
lantia and Severinia which do not belong to the True Cit-
rus Fruit Trees were distant from other accessions;(2)
Microcitrus and Eremocitrus clustered together , which
were close to Citrus;(3)Three trifoliate orange acces-
sions;(4)Fumin trifoliate orange and C.halimii;(5)
C.aurantium , C.sinensis C.maxima and C.paradisi;
(6)C.honghensis;(7)C.ichangensis and C.latipes;
(8)Two Fortunella accessions;(9)three mandarin ac-
cessions and C.tachibana;(10)the cluster consists of
two subclusters , one subcluster comprises of C.lemonia ,
C.jambhiri and C.medica , C.limon while the other
comprises of C.aurantifolia var.mexican lime , C.au-
rantifolia and C.hytrix.
Fig.2　A neighbour-joining tree for 29 accessions of Citrus and its related genera derived from bootstrap
analysis(500 relications)of SSR data with percentage of confidence levels higher than 40% on arms
84 遗　传　学　报 30 卷　
3　Discussion
In the present study , based on the results of SSR
analysis ,Microcitrus was nested in Citrus , which implied
the close relationship between Microcitrus and Citrus.The
results was in agreement with the morphlogical studies of
Swingle and Reece[ 2] and isozyme results[ 15] .Likewise
complete pairing of chromosomes in meiosis in the inter-
generic hybrid between Microcitrus and Citrus demonstrat-
ed that they originated from a common ancestor
[ 16] .These
data indicates that it is acceptable as proposed by Mab-
berley[ 17] to reclassify Microcitrus into Citrus.
Huang
[ 18]
postulated that Poncirus was probably the
offspring of certain Citrus species , which evolved into
Poncirus when it was spread from the south to the north of
China.However , Poncirus was believed to be phylogenet-
icly distant from Citrus in most studies
[ 15 , 16 ,19 ,20] ,which
was similar in the SSR results herein.Therefore it is hard
to consider Poncirus was derived from Citrus.
Fumin trifoliate orange was found by Ding et al.[ 21]
in 1977 in Yunan province.Based on the morphological
data ,Ding et al recognized it as a new species Poncirus
polyandra S.Q.Ding et al.., which was subsequently
supported by isozyme data
[ 22]
and pollen morphology
[ 23] .
However Guo et al.[ 24] asserted it was the intergenric hy-
brid between Citrus and Poncirus.Based on the isozyme
data , Fang postulated that the taxa with the higher ho-
mozygosity was of more primitive origin
[ 25] .The distinct
position of Fumin trifoliate orange was reflected by the fact
that it was homozygous in six of seven SSR loci ,whereas
the three P .trifoliate accessions had only three homozy-
gous loci.Thus , our results gave further evidence to the
species status of Fumin trifoliate orange.
Swingle and Reece[ 2] reported that there were signif-
icantly differences between Fortunella and Citrus in mor-
phology.Of the six genus of the True Citrus Fruit Trees ,
Fortunella was the most simple and primitive genera while
Citrus represented the highest stage of evolution.Howev-
er , recent molecular approaches found there has not the
same level of divergence at the molecular level[ 26] .It was
not unexpected since morphological change and molecular
divergence are quite independent , responding to different
evolutionary pressures and following different rules[ 27] .
Fortunella was nested within Citrus based on isozyme[ 28] ,
RFLP[ 26] and protein analysis[ 29] .But it was found to be
distant from Citrus by cpDNA data
[ 20 , 30] ,which is in ac-
cordance with the present research.This incongruence may
result from different molecular marker sampling different
regions in the genome
[ 31] ,which manifests that it is nec-
essary to analyze with different methods dealing with Cit-
rus phylogeny.
The research demonstrated that C.honghensis was
related to pummelo , which was reported by RFLP and
RAPD data elsewhere[ 26] .The numerical taxonomic study
by Zhong and Ye[ 32] also showed pummelo had some rela-
tionships with C.honghensis.C.hystrix is a confusing
type.Swingle and Reece[ 2] believed it was a good
species.It was clustered together with C.medica by
RAPD data[ 33] , so did by our SSR data , implying possible
relationship between C.hystrix and C.medica.Zhou[ 34]
also deduced that it might be of hybrid origin based on the
cladistic analysis of morphological characters.However , its
heterozgyosity index based on RFLP was very low
[ 26] ,
which argued against the view of hybrid origin.
The Papeda did not form a single cluster , in agree-
ment with RFLP data[ 26] , cpDNA data[ 20] ,numerical taxo-
nomic study of morphological characters
[ 32] .However , the
RFLP plus RAPD[ 26] RAPD data[ 20] clustered Papeda to-
gether.The close relationships between Citrus and Papeda
might lend the evidence to Tanaka s hypotheses that Cit-
rus originated in Asia about 30 million years ago from
Papeda species[ 35] , which is consistent with cpDNA re-
sults[ 20] .
Recently , with the application of isozyme , RAPD ,
RFLP and ISSR , the focus of research on Citrus classifica-
tion has shifted from the speices number to the phyloge-
netic relationships.The origin of several biotypes has been
clarified
[ 6] .More and more studies supported the view-
point of three basic species , however the three basic
species do not involve Papeda while several studies found
the strong relationship between Papeda and Cit-
rus[ 15 , 20 ,26 ,28 , 36] .The present data did not support the
subgenus separation as put forward by Swingle and
Reece[ 2] or Tanaka[ 3] and Papeda should have played an
important role in the origin of Citrus.Consequently , it
85　1 期 庞晓明等:用 SSR标记研究柑橘属及其近缘属植物的亲缘关系
should be interesting to study more samples from Papeda
accessions , such as C.ichangensis , C.hystrix and C.
macrosperma[ 37] , in order to further decipher the Citrus o-
rigin and phylogenetic relationships.
Acknowledgements:The authors wish to thank Prof.ZS
Chen ,Mr.D Jiang ,Mr.XQ Li of Citrus Research Institute
of Chinese Academy of Agricultural Science for their help
in collecting the materials.Further more ,we would like to
extend our appreciation to Dr.DQ Fang for donating the
DNA of three accessions and Dr.JH Liu and Dr.XP Wen
for their critical reviewing the manuscript.
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(责任编辑:周　素)
“纪念 DNA模型发表 50周年暨《遗传学报》创刊 30周年
学术研讨会”报名通知
　　1953 年 4月 25 日 , 英国《自然》杂志发表了沃森和克里
克的文章“核酸的分子结构———脱氧核糖核酸的一个结构模
型” 。 DNA 双螺旋结构模型的建立 , 标志着人类在揭示生命
的遗传奥秘方面迈出了具有里程碑意义的一步。
《遗传学报》是中国遗传学会和中国科学院遗传与发育
生物学研究所主办 、科学出版社出版的高级学术刊物 , 中国
自然科学核心期刊 , 国家重点期刊 , 全国期刊方阵“双百期
刊” ,已被美国化学文摘(CA)、生物学文摘(BA)、医学索引
(MED)等 30余种国内外重要检索系统和数据库收录。
为了纪念 DNA 模型发表 50 周年暨《遗传学报》创刊 30
周年 ,特举办本次大型学术研讨会。主题是:从 DNA 模型到
基因组。
1.主办单位:中国遗传学会 , 中国科学院遗传与发育生
物学研究所 ,东南大学
承办单位:《遗传学报》 、《遗传》杂志编辑室 ,东南大学
医学院遗传学研究中心
2.大会主席:中国遗传学会理事长　赵寿元
《遗传学报》 、《遗传》杂志主编　朱立煌
东南大学校长　顾冠群
大会副主席:《遗传学报》副主编　陈　竺　吴常信
李家洋　张启发
中国科学院遗传与发育生物学研究所
副所长 薛勇彪 、东南大学副校长 浦跃朴
大会秘书长:李绍武 、谢　维
3.会议日期:2003年 4 月 20～ 24 日
2003年3 月20 日前报名并交费者, 注册费 400元 ,会上交
款者 ,注册费600 元。国内公司展费 2000元 ,外商 3000元。
4.地　　点:南京丁家桥:东南大学医学院
5.会议内容:(1)纪念 DNA双螺旋结构模型发表 50 周年
学术报告 , 优秀征文颁奖
(2)《遗传学报》 30 年回顾与展望;表彰优秀编委;推荐下
届编委人选
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家 、热心读者 、审者 、大会征文作者 、生命科学科相关领域科
研 、开发与教学工作者及研究生等。
7.征文范围:遗传学 、发育生物学 、基因组学 、分子进化
等领域有创新性的研究论文。截止日期:2003年 3 月 30 日。
征文一式两份 ,注明“会议征文” ,免收审稿费。收稿后及
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表 ,安排作大会报告 ,从中评选优秀论文 ,颁发证书和奖金。
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学历 、职务 、职称 、单位 、地址 、邮政编码及电子信箱等。 2003
年 3 月发报到通知。
8.报名方式:
地址:北京市安定门外大屯路 917 大楼　《遗传学报》编
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E-mail:swli@genetics.ac.cn;网上报名:www.Chinagene.org
《遗传学报》编辑部
2003年 1 月 10 日
87　1 期 庞晓明等:用 SSR标记研究柑橘属及其近缘属植物的亲缘关系