全 文 ：广 西 植 物 Guihaia 27(1)：44— 47 2007年 1月
Identificati0n of closely related mango
cul tivars by ISSR
HE Xin-HuaI，2，LI Yang—RuiI ，GUO Yong—Ze2，
OU Shi-J in2．LI Rong-BaiI
(1．Laboratory of Guangxi Crop Genetic Improvement and Biotechnology，Guangxi Academ y of Agricultural Sciences，
Nanning 530007，China；2．College of Agriculture，Guangxi University，Nanning 530004，China)
Abstract：Seven carabao mango cultivars or lines and Liuzhou lusong mango(JVlangifera indica Linn)were examined
with ISSR primers．Of the 3O primers screened，6 primers gave reproducible，polymorphic DNA amplification pat—
terns，and were selected to construct a DNA fing erprinting map to distinguished carabao mango cultivars or lines．Ac-
cording to the banding patterns，al cultivars t~ted in this study were distinguished from each other by every one of 6
selected primers and showed ample genetic diversity，indicating that ISSR-PCR was an effective method for cuhivar i-
dentification of mango cultivars and lines．Based on UPGMA analysis of 69 selected bands，the carabao showed the
low~t similarity to al other cultivars while Gaozhou carabao，Zhanjiang carabao，Tianyang xiangmang ，Jinqian—
mang，Liuzhou lusong，Yuefimang No．1 and Panxi red carabao could be clustered into one group．
Key words：mango(Mangifera indica)；ISSR；cultivar identification
CLC number：Q943，Q949 Document code：A Article ID：1000—3142(2007)01—0044—04
Molecular markers provide an attractive and more
reliable alternative to morphological markers．In mango
(Mangifera indica IAnn)，phenotypic ma rkers have been
the most common method for describing cultivars．A1一
though this approach is useful to distinguish dismmly re—
lated cultivars，its reliable application ha s proven more
dificulties when it comes to differentiating closely relat—
ed lines or off types of a particular cultivar．Therefore，a
more refined technique，such as molecular ma rkers that
are highly polymorphic，is required．
In the 1ast decade，ma ny molecular methods have
been implemented in ma ng o，but random amplified poly—
mo~hic DNA(RAPDs)(Bally et a1．，1996；Deng et a1．，
1999；Karihaloo et a1．，2003；Lopez-Valenzuela et a1．，
1997；Ravishankar et a1．，2000；Schnel1 et a1．，1992，
1995；Xu eta1．，1998)and amplified fragment length po l—
ymorphism(AFIPs)(Eiadthong et a1．，2000 Fang et
a1．，1999，2000，2001)were more commonly used，and the
two methods have also ben applied in discrimination of
ma ng o cuhivars in China(Deng eta1．，1999；Fang eta1．，
1999，2000，2001；Xu et a1．，1998)．Among these molecu—
lar methods，inter-simple-sequence-repeats (ISSRs)is a
relatively novel technique and has ben proven to be a
powerful，rapid，simple and inexpensive way to assess ge—
netic diversity(Fang et a1．，1997a)or to identify closely
related cultivars(Fang et a1．，1997b)and to study evolu-
tionary processes in tree species(Wolfe et a1．，1998)．IS—
SRs have ben applied in identification of ma ngo in 一
land(Eiadthong et a1．，1999)and in Australia(Gonzalez et
a1．，2002)，but ha ve not been reported in na yet．
Carabao introd uced from Philippines was widely
planted in our ma ng o cultivation areas，and ma ny new
1ines have been selected from carabao for severa1 dec-
ades，While there is much confusion and difficulty in i—
Received date：2005-‘10-‘10 Accepted date：2006-01—26
基金项目：广西自然科学基金(桂科自0542022)；广西农科院博士后基金 [Supported by Natural Science Foundation of Guangxi
(0542022)；Postdoctoral Foundation of Guangxi Academy of Agricultural Sciences]
作者简介：何新华(1966-)，男，湖南衡阳人，博士，教授，研究方向：果树生物技术。
‘通讯作者(Author for correspondence)
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1期 何新华等：ISSR鉴定亲缘关系非常近的芒果栽培品种 45
dentifying the existing carabao cultivars in China．Thus，
in the present study，ISSRs were used to discriminate ca—
rabao cultivars or lines．
1 M aterials and methods
1．1 Plant materials
Gaozhou carabao，Zhanjiang carabao，Tianyang
xiangmang(Seedling Variation of Carabao)，Jinqianmang
and Liuzhou lusong (variation of Philippine mango varie-
ty)from the germplasm colections of Guang xi Academy
of Agricultural Sciences，and Carabao，Yueximang No．1
(Seedling ation of Ca rabao)and Panxi red carabao
(Seedling Variation of Carabao)from the germplasm col—
lections of Subtropical Plant Institute of Guang~ were
employed as the plant materials in the present study．
1．2 DNA extraction
DNA was extracted from ma ngo leaves using the
Cetyl Trimethyl Ammonium Bromide(C【 )method
described by Doyle and Doyle(1990)with a little modi—
ficatior- Leaf samples of 0．5 gwere groundtofine p
er in liquid nitrogen and added into a 10 mL tube with 4
mL preheated (65℃)extraction buffer consisting of 2
CTAB，1．4 mol／L NaC1，0．1 (v／v)~-mercaptoetha—
nol，20 mmol／L口了IA，100 mmol／L is—HCl(pH8．
0)，and 1 (w／v)PVP-40．The homogenates were incu—
bated at 65℃ for 1 h and extracted one time wi th 4 mL
chloroform：isoamyl alcohol(24：1)solution．The tubes
were mixed for about 5 mi n，and centrifuged at 12 000
rpm for 10 mi n． The supernatant was transferred into a
new tube containng twice volume of 100 ethanol and
i／i0 volum e 3 mol／L NaAc，mixed gently，left at一20℃
for lh and spun at 12 000 rpm for 15 mi n． 硒 e supema—
tant was then discarded and the pellet washed twi ce with
70 ethano1．The pe let was dried at room tempera-
ture，and resuspended into 200止 0．1XTE with RNase
A 1、he DNA was precipitated wi th 400ttL 100 etha -
nol at一20℃ for 1 h an d centrifuged at 12 000 rpm for
15 mi n． The pellet was resuspended into 100 L 0．1X
TE．The DNA concentrations were detected by Eppen-
dof Biophotometer．
1．3 ISSR analysis
ISSRs primers were designed by ourselves or re-
ferred to the papers of Hadthong et a1．(1999)and
Gonzalez et a1．(2002)and synthesized by Shanghai
Sangon Biological Engineering Technology & Services
Co．，Ltd Thirty primers wexe screened in the present
study．Each 20 L amplification reaction consisted of 10
mmol／L s—HCI(pH8．3)，50 mmol／L KC1，1．5 mmoll
L MgCI2，0．2 mmol／L mixed dNTP，0．25 ttmol／L prim-
er，1 unit rTaq polymerase(Takara Biotechnology ，Ja—
pan)，and approxima tely 60 ng genomi c DNA．Amplifi—
cation was perform ed in Thermcyder under the follow-
ing conditions ：5 mi n at 94℃ for 1 cycle，folowed by 1
mi n at 92℃ 。1 mi n at 47℃ and 2 mi n at 72℃ for 40
cycles．and 10 mi n at 72℃ forfinal extens ion． The am-
plifcation products were loaded onto a 2．0 agarose gel
1 XTBE buffer for electrophoresis at 100 V for 1 h，
and then visualized by stainng the gel wi th ethidium
bromi de e size of each fragment was estima ted wi th
reference to C-eneRulervM 100 bp DNA ladder plus
(Ⅳ口BI)．
I．4 Data analysis
Hghty reproducible bands from selected primers
were scored as 1(presence)or 0(absence)for 8 cultivars
or lines tested．Then，the unweighted pair-group method
using arithmetic averages(UPGMA)cluster analysis was
perform ed using NTSYSpc version 2．1e software(Nu—
merical Taxonomy System version 2．01)．
2 Results and analysis
2．1 Screening of primers and diversity analysis of
man go genomic DNA
Of the 30 ISSR primers screened。6 primers(Table
1)were selected in our ana lyses for their reproducible
and polymorphic DNA am plifcation patterns(Hg．1)．
1、he P(’R by 6 ISSR primers yielded a total 80 bands and
69 scorable polymorphic ma rkers(Table 1)，accounting
for 86．3 of tota1．Ea ch primer could produce 7 to 16
DNA polymorphic bands，and the size of bands amplified
was between 200 bp to 2 800 bp．
According to the banding patterns obtained wi th 6
selected primers，all carabao cuhivars tested in this study
could be distinguished from each other，indicating that
ISSR—f℃R was an effective method for cultivar identifi—
cation of mango，and the genetic diversity of tarabao co]一
tivars or lines was clarified by comparing 69 polymorphic
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46 广 西 植 物 27卷
loci(Table 1)．
Table 1 Sequence of reliable ISSR primers and the
number of scorable polymorphic bands of each primer
Note： “UBC”was stood for primers designed by Biotechnology
Laboratory，University of British Columbia，Canada~“GXU”repre·
sented primer designed by ourselves
2．2 Cluster analysis
The dendrogram obtained by the UPGMA cluste-
M 1 2 3 4 5 6 7 8
3000
2000
1 500
1 200
1031
900
800
700
600
500
400
300
200
100
ring method revealed the genetic relationship of 8 mango
cuhivars tested in this study(Fig．2)．On the dendro—
gram ，the carabao showed the lowest similarity to all
other cultivars while Gaozhou carabao，Zbanjiang eara—
bao，Tianyang xiangmang，Jinqianmang，Uuzh0u lusong，
Yueximang No．1 and Panxi red carabao could be clus—
tered into one group．
3 Discussion
In the previous papers，it was told that the Gaozhou
carabao and Zbanjiang carabao are the same cuhivar with
different names as carabao introduced from Philippine，
and Tianyang xiangrna~ ，Jinqianmang，Yueximang No．1
and Panxi red carabao are lines or cultivars selected from
carabao。so there is much confusion and difficulty in i—
dentifying these carabao
M 1 2 3 4 5 6 7 8
Fig．1 PCR amplifed patterns by UBC-840(Hg．A)and UBC-841(Fig．B)primer in eight carabao cultivars,
Lane M：GeneRulerVM100 bp DNA ladder plust Lime 1-8；Represented for cultivers Gaozhou carabaotghanjhng carabao，
Tmnyang algmang·Jinqianmang，Liuzhou lusong，CarabaotPanxi red carabao，and Yueximang No．1 respectively．
Gaozhou
Zhanj i ang
Ti anYang
di nai an
Li uzhou
Yuexi
Panxi
Cai-abao
0．52 0．60 0．68 0．76 0．84
Coeff i c i ent
Fig．2 Dendrogram of phylogenetic relationship
among 8 carabao cultivars or lines by ISSR
markers based on UPGMA analysis
confusion and dificulty in identifying these carabao cul—
tivars by morphological nlaIkers．But they showed dis—
tinct DNA amplification patterns and high polymorphism
byISSRwith one 0fthe 6 selected primersinthepresent
study，it was very easy to distinguish them．ISSR tech-
nique in this study see玎lS to be quite valuable for identi-
fication of carabao mango cultivars or lines an d evalua—
tion of their genetic diversity．
The result indicated that the carabao showed the
lowest sim larity to the other cultivars in the study，the
reasons may be as follows：(1)the carabao in the study
mi ght not be the true carabao originated in Philippines
(2)the carabao in the study could be mutant line of tara—
bao from Philippines because bf top-grafting and envi—
ronmental factors．
The Liuzhou lusong orignated in Philippines was a
cultivar diferent from carabao，but it showed high simi—
larity to other carabao cultivars or lines in the present
0 0 0 0 3 0 0 0 0 0 0 0 0 0 ∞{三12 98 76543 2，
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何新华等：ISSR鉴定亲缘关系非常近的芒果栽培品种 47
study，especialy the similarity between Huzhou lusong
and Yuemmang No．1 were more than 7O 9／6，indicating
tha t it ha d close relationship with other carabao culti—
vats．Therefore，Liuzhou lusong could bdong to fl culti—
vat or line of carabao type．
Acknowledgements 肠 e authors gratefully tha nk
Prof．Peng Hong-Xiang ，Ms Ren Hui of Horticultural
Institute，Guangxi Academy of Agricultural Sciences；
Associate Prof．Huang Guo-Di of Subtropical Plant In—
stitute of Guangxi；Associate Prof．Tang Zhi-Peng of
Guangxi University for supplying the plant materials．
●
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ISSR鉴定亲缘关系非常近的芒果栽培品种
何新华 一，李杨瑞h ，郭永泽2，欧世金2，李荣柏1
(1．广西农业科学院 广西作物遗传改良生物技术重点开放实验室，南宁 530007；2．广西大学 农学院，南宁 530004)
摘 要：用 ISSR技术鉴定7个吕宋芒品种(系)和柳州吕宋芒。从30个引物中筛选出6个多态性好的ISSR引物建
立DNA指纹图谱用于区分吕宋芒品种(系)。分析DNA指纹图谱，发现这 6个引物中每个引物都能区分吕宋系列
品种(系)，表明 ISSR-PCR技术对芒果品种(系)的鉴定非常有效，能区分亲缘关系很近的品种(系)。基于 69条多态
性条带的聚类分析结果t发现吕宋芒和其它供试的 7个品种(系)同源性低，而这 7个品种(系)：高州吕宋芒、湛江吕
宋芒、田阳香芒、金钱芒、柳州吕宋芒、粤西一号、攀西红吕宋同源性较高，可归为一类。
关键词：芒果；ISSR；品种鉴定
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