全 文 ：利 用 ISSR 分 析 紫 云 英
（Astragalus sinicus L.）品种遗
传多样性
张慧，陈济琛，林新坚 * （福建省农业科学院
土壤肥料研究所，福建福州 350013）
摘 要 株系紫云英品种的 DNA 为模板进行
PCR 扩增，筛选出 40 条扩增条带较好的 ISSR
引物， 其中有 10 个引物扩增出的条带多态性
较好，从 500~3 000 bp 共扩出 684 个条带，平
均多态率 59.2%，表明供试的紫云英品种资源
的遗传多样性相依程度较高。采用 NTSYS 2.1
软件分析，22 个紫云英品种间的相似性系数
界于 0.63~0.95。 UPGMA 法聚类将 22 个紫云
英品种分为 4 类， 研究表明 ISSR 分子标记方
法可较好的应用于紫云英品种遗传多样性分
析上，并为紫云英品种间的鉴定分类提供了分
子水平的依据。
关键词 紫云英； ISSR 引物；遗传多样性
基 金 项 目 农 业 部 公 益 性 行 业 专 项
（201103005）。
作者简介 张慧（1981- ），女，吉林安图人，助
理研究员， 从事土壤微生物方面研究，E-mail：
zhanghui08@126.com。*通讯作者，研究员，从事农
业微生物方面研究，E-mail：xinjianlin@163.net。
收稿日期 2014-05-16
修回日期 2014-08-01
Genetic Variability of Astragalus sinicus L. Based
on ISSR Markers
Hui ZHANG, Jichen CHEN, Xinjian LIN*
Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
Supported by the Public Benefit Research Foundation of National Departments, China
(201103005).
*Corresponding author. E-mail: xinjianlin@163.net
Received: May 16, 2014 Accepted: August 1, 2014A
Agricultural Science & Technology, 2014, 15(8): 1295-1298
Copyright訫 2014, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
C hinese milk vetch (Astragalussinicus L.) belongs to theLegume Astragalus, has been
cultivated in China, Japan, Vietnam,
Korean Peninsula and other countries
for thousands of years, and popular-
ized to wide distributions. Chinese milk
vetch is mainly used as one of green
manure crops, nectar plants and
feeds, and it plays an active role in en-
vironmental protection[1-2]. With the on-
going development of social economy
and the improvement of people’s liv-
ing standards, the current society has
proposed higher request for tillage
quality, food security, ecological envi-
ronment and high quality agricultural
products, which would restore the
plantation and usage of green manure
and bring about great benefit and
opportunities.
A. sinicus varieties cannot adapt
to the requirement of modern agricul-
tural development because of their
long time breeding lag, and the pro-
duction of varieties which are reduced,
old, mixed and many other problems.
It is difficult to accurately identify the
genotypes of A. sinicus by using tradi-
tional variety evaluation such as mor-
phological characteristics and obser-
vation of growing features. To date, an
efficient molecular marker technology
has become the preferable method to
solve this problem rapidly [ 3 -5 ] . Inter -
simple sequence repeat (ISSR) tech-
nology is a dominant molecular mark-
er[6], and it also has been widely used
to estimate nucleotide sequence varia-
tions in different plants[7-8].
ISSR was initiated in 1994 with
many advantages such as simple op-
eration, low cost, high sensitivity, high
polymorphism, low quantity of DNA
and the stability of simple sequence
repeats (SSRs)[9-10]. There are no avail-
able references for evaluating genetic
diversity of Chinese milk vetch by IS-
SR markers. To prevent the milk vetch
species extinction, mixed, the trend of
degradation, 22 A. sinicus varieties, in-
cluding the Fujian purple series and
the series from the national crop
germplasm repository, were used to
evaluate the genetic relationship and
diversity of A. sinicus accessions. To
some extent, it provides a theoretical
basis for future germplasm conserva-
tion, evaluation, utilization, genetic
map construction and Chinese milk
Abstract Genetic relationships among 22 accessions of Astragalus sinicus L. collect-
ed from different provinces of China were analyzed by inter-simple sequence repeat
(ISSR) markers. The results showed that 10 highly reproducible ISSR fragments a-
mong 40 primers were screened. Using these primers, a total of 684 ISSR frag-
ments from 500 to 3 000 bp were amplified, and 59.2% of them showed polymor-
phic by unweighted pair-group method with arithmetic (UPGMA) analysis. It revealed
that the 22 accessions had a similarity range from 0.63 to 0.95, and existed biolog-
ical diversities. Based on cluster and principal coordinate analyses, all accessions
could be divided into four distinct groups.
Key words Astragalus sinicus L.; ISSR; Genetic diversity
Agricultural Science & Technology 2014
Table 1 Geographical origins of the 22 Astragalus sinicus L. accessions
Accession Origin (Province) Accession Origin
A01 Liling, Hunan A12 Zhejiang Academy of Agricultural Sciences
A02 Dawu, Hubei A13 Zhejiang Academy of Agricultural Sciences
A03 Xiangxiang, Hunan A14 Fujian Academy of Agricultural Sciences
A04 Yidu, Hubei A15 Fujian Academy of Agricultural Sciences
A05 Hunan Academy of Agricultural Sciences A16 Fujian Academy of Agricultural Sciences
A06 Guangxi Academy of Agricultural Sciences A17 Fujian Academy of Agricultural Sciences
A07 Ningxiang, Hunan A18 Fujian Academy of Agricultural Sciences
A08 Bingku, Japan A19 Fujian Academy of Agricultural Sciences
A09 Hunan Academy of Agricultural Sciences A20 Fujian Academy of Agricultural Sciences
A10 Ningbo Agricultural Research Institute A21 Fujian Academy of Agricultural Sciences
A11 Ningbo, Zhejiang A22 Xiangxiang, Hunan
vetch breeding programs.
Materials and Methods
Plant materials
The seeds of the total of 22
A. sinicus varieties were collected for
the tests (Table 1). Seeds were wetted
on moisture filter paper and germi-
nated for 12 h, and then sown in 10
cm diameter pots, three seeds each
pot with triplicate. The seeds were
cultivated at room temperature (22 -
25 ℃) in laboratory; after three weeks,
the seedlings were used for DNA
extraction.
DNA extraction
Total genomic DNA was extracted
from 15 g of fresh seedlings by using
rapid isolation Kit of Extensive Ge-
nomic DNA for plants (Biomed
Biotechnology, Beijing, China). DNA
quality and concentration were
checked with lambda DNA standards
on 1.0% (w/v) agarose gel. The ex-
tracted DNA was stored at -20℃ after
diluted to 50 ng/μl in order to be used
in amplification by polymerase chain
reaction (PCR).
Primer screening and PCR amplifi-
cation
The 40 primers supplied by
Shenggong Inc. (Shanghai, China)
were screened for their ability to ampli-
fy ISSR markers, and 10 of these
primers were selected for the present
study based on their reproducibility,
clarity, and highly polymorphic nature
of product bands (Table 2). Each PCR
amplification was performed in a final
volume of 25 μl reaction mixture, con-
taining 50 ng of template DNA, 0.2
μmol/L primer and 12.5 μl of 2 × Taq
PCR Master Mix (TaKaRa Biotech-
nology, Dalian, China). PCR amplifi-
cations were performed as the follow-
ing: an initial 5 min denaturation at
94 ℃ following by 35 cycles of 1 min at
94 ℃ , 1 min at 56 ℃ , 1 min at 72 ℃ ,
and a final 7 min extension at 72 ℃ .
PCR products were separated by
electrophoresis on 2.0% (w/v) agarose
gel at a constant voltage (90 V) for ap-
proximately 50 min. GoldView (0.5
μg/ml) was added for visualization with
UV light. Clearly and reproducibly dis-
tinguished bands were recorded and
used in the following analysis.
Data analysis
Each ISSR fragment was scored
as present (1) or absent (0) for each of
the 22 DNA samples, excluding the
weak and blurred bands, thus gener-
ating a binary data matrix. A locus was
considered to be polymorphic if more
than one band at the same position
was detected for all the materials. The
relative genetic dissimilarity was esti-
mated according to Nei (1979)[11] and
calculated based on SIMQUAL mod-
ule similarity coefficient. Cluster anal-
ysis was undertaken with the numeri-
cal taxonomy and multivariate analysis
system (NTSYS) program, version 2.1
(Exeter Software, Setauket, NY), in
accordance with UPGMA found in
SAHN module of the NTSYS program.
Based on genetic similarity, principle
coordinate analysis (PCA) was under-
taken to estimate the genetic dis-
tances among the major groups using
the DCENTER and EIGEN modules of
the NTSYS program. The 3D scatter
plots were generated and compared
with the dendrogram.
Results and Analysis
Polymorphism of the ISSR markers
In this study, 10 from a total of
100 primers showed clear and repro-
ducible bands. These 10 primers were
then used to analyze genetic diversi-
ties of the 22 accessions (Fig.1). A to-
tal of 684 fragments ranging from 500-
3 000 bp were amplified with an aver-
age of 68.4 bands per primer, among
which 405 (59.2%) were polymorphic
from ISSR analysis (Table 2). The
numbers of reproducible and polymor-
phic bands produced from various
primers were different. The highest
number of amplification products was
obtained with the primer UBC834, and
the lowest with UBC873. The average
Lanes 1-22 represent samples of A01-A22, referring to sample information in Table 1.
Fig.1 Representative agarose gel displaying sequence variation among Astragalus sinicus
L. samples revealed by the technique of ISSR using primer UBC836
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Agricultural Science & Technology2014
Table 2 Length of the amplification products and polymorphism detected by 10 ISSR primers among the 22 Astragalus sinicus L.
accessions (R=A, G; Y=C, T).
Primer code Sequence (5’-3’) Length ofamplified band
Annealing
temperature∥℃
Number of
amplified band
Number of
polymorphic band
Percentage of
polymorphic
band∥%
UBC807 (AG)8T 600-1 900 49.0 81 34 41.98
UBC809 (AC)8G 750-2 200 56.0 82 48 58.54
UBC834 (AG)8YT 650-1 900 54.0 96 28 29.17
UBC836 (AG)8YA 600-1 700 55.8 68 46 67.65
UBC844 (CT)8RC 650-1 900 52.3 82 38 46.34
UBC856 (AC)8YA 750-2 300 53.0 53 53 100.00
UBC866 (CTC)6 800-2 000 48.6 77 57 74.03
UBC873 (GACA)4 500-900 56.4 40 18 45.00
UBC881 (GGGTG)3 900-1 600 53.0 49 27 55.10
UBC889 DBD(AC)7 750-3 000 53.0 56 56 100.00
Total - - - 684 405
Average - - - 68.4 40.5
number of bands among the 10
primers was 68.4 and the number of
polymorphic fragments for each primer
varied from 18 to 57 with an average of
40.5.
Analysis of genetic diversity
The genetic similarity coefficients
(GSCs) varied between 0.63 and 0.95
with an average of 0.79 among the 22
accessions. The lowest GSCs (0.63)
were detected between A20 and oth-
ers. Hence, these were the least -re-
lated accessions, whereas the highest
GSC was 0.95 detected between ac-
cessions A18 and A19, indicating that
a very close relationship existed be-
tween these two accessions.
A dendrogram of these acces-
sions was drawn by using Cluster
analysis with the UPGMA method.
Twenty-two accessions were empiri-
cally divided into four major groups (A,
B, C and D; Fig.2) at the 0.66-similari-
ty level. Group A included only one ac-
cession (A20); Group B contained four
(A5, A21, A6, A22); Group C con-
tained three (A2, A7, A11); Group D in-
cluded the remaining 14 (A1, A10, A3,
A14, A12, A9, A15, A13, A4, A8, A16,
A17, A18, A19).
Twenty-two accessions were
classified by principle coordinate anal-
ysis (PCA) into four distinct groups
(Fig.3), namely Groups A, B, C, and D.
PCA was consistent with the UPGMA
cluster analysis. Figure 3 showed the
distribution of different accessions
based on two principal axes of varia-
tion using PCA assay. The percent-
ages of PC1 and PC2 variations were
17.2% and 14.9%, respectively.
Discussion
There were 111 germplasm re-
sources (varieties) of A. sinicus which
had been collected and preserved in
the laboratory since 2006. Among of
these, 22 varieties were used in this
research. Characterization of A. sini-
cus germplasm is important to assess
breeding programs. Previous reports
indicated that ISSR markers were
useful and highly variable for
germplasm analysis in plants, such as
Jatropha curcas and Sea buckthorn
(Hippophae L.) [10, 12], but there was no
literature reports using ISSR markers
to analyze A. sinicus. Ten ISSR
The accession numbers are the same as those listed in Table 1.
Fig.2 Dendrogram showing relationships among different Astragalus sinicus L. accessions
using UPGMA analysis
Fig.3 Principle coordinate analysis (PCA) of
the 22 Astragalus sinicus L. acces-
sions based on the genetic similarity
matrix derived from the ISSR marker
analysis
1297
Agricultural Science & Technology 2014
Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WU
primers were selected to assess the
genetic diversity of A. sinicus and pro-
duced 684 characteristic bands with a
polymorphic level of 59.2%.
Based on the dendrogram, all ac-
cessions of A. sinicus were divided in-
to four major clusters at the similarity
level of 0.66. The polymorphism indi-
cates that DNA of A. sinicus has its
mutation probability. In addition, ran-
dom primers have high potential for
detecting polymorphisms at the
species level. The dendrogram gener-
ated from the UPGMA of Jaccard’s
similarity. PCA assay was shown in
broad agreement with dendrogram
and with the accepted taxonomy. Four
main groups were clustered (Groups
A, B, C and D), and the most closely
related accessions were grouped.
The ISSR method has been re-
ported to be more reproducible [13] and
produces more complex marker pat-
terns than the RAPD approach [14 ] ,
which is advantageous for differentia-
tion in numerous plant species, in-
cluding rice[15], castor bean[16] and mul-
berry [17]. The identification of parents
with high genetic variability has been a
goal of many breeding programs that
aim to explore the heterosis. Thus,
germplasm characterization is neces-
sary to provide information on gene
sources for future use and to prevent
the loss of resources. The ISSR mark-
ers display polymorphism among na-
tive species of A. sinicus in China.
Polymorphism levels in this study also
suggest that ISSR markers are a reli-
able and available tool to identify ge-
netic diversities of native A. sinicus
species.
Acknowledgments
The authors are grateful to Prof.
Mingkuang Wang for his critical review
of this manuscript.
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