全 文 ：Analysis of Genetic Relationship among
Medicago sativa, Medicago falcate and Trigonella
foenum-graecum Using ISSR
Lei LIU1, 2, Zongli WANG1, Zhiyong LI1*, Guodong ZHOU1, Wengui SHI1, Hongyan LI1, Liyan CAI1
1. Grassland Research Institute, CAAS, Hohhot 010010, China;
2. Graduate School, CAAS, Beijing 100081, China
Supported by Forage Germplasm Resource Protection Project of Ministry of Agriculture,
China.
*Corresponding author. E-mail: zhiyongl@public.hh.nm.cn
Received: June 20, 2012 Accepted: August 30, 2012A
Agricultural Science & Technology, 2012, 13(10): 2076-2079
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
Abstract [Objective] This study aimed to analyze the genetic relationships among
Medicago sativa, Medicago falcata and Trigonella foenum-graecum. [Method] ISSR
technique was adopted to determine their genetic relationships. [Result] M. sativa,
M. falcate and T. foenum-graecum had a broad genetic base. T. foenum-graecum
shared closer relationship with M. falcata rather than M. sativa. The study on rela-
tionship between M. sativa and T. foenum-graecum was advantageous to identify
disputable transition types. But a boundary should be found to identify species to be
M. sativa or T. foenum-graecum. [Conclusion] This study will provide reference for
identifying some disputable transition types.
Key words Medicago sativa L.; Medicago falcata L.; Trigonella foenum-graecum L.;
Relationship; ISSR
T rigonella foenum-graecum L.also known as remote lemon-grass herb and Fenugreenk, is
an annual herb in Faboideae of Legu-
minosae[1]. Among the nine Trig-onella
species in China, four are wild species,
including T. cancellata Desf, T. mo-
nantha CAMeyer, T. orthoceras Kar. et
Kir, and T. arcuata CAMeyer, and they
are distributed only in Xinjiang[2]. Some
transitional species which are often re-
ferred to as Medicago id or Trigonella
id in nature are mainly distributed
Pamir, Kashmir, Hindu Kush in Central
Asia, and China’s western highlands
and their surrounding mountains (in
Qinghai, Xinjiang, Tibet, Yunnan and
other provinces). The pods of these
transitional species are flat and long,
or crescent or wide sickle-shaped, dif-
ferent from the pods of typical
Trigonella and Medicago species.
These transition types were once sep-
arated as Melilotoides Heister ex Fabr.
or Turukhania Vass.[3-4], but some sch-
olars classified them into Trigonella[5-7]
orMedicago[8-9].
ISSR molecular marker technolo-
gy has better stability and polymor-
phism and has been successfully used
in the studies about genetic relation-
ship of many plants[10], such as Primula
malacoides [11], Cymbidium [12], Oryza
sativa [13], Mangifera indica [14], Nicotiana
tobacum [10, 15], ornamental Fritillaria [16],
Prunus mume[17], Glycine max[18], Cord-
yceps sinensis [ 19 ] , Luffa cylindrica [ 20 ]
and Camellia sinensis[21].
ISSR molecular marker technolo-
gy was used in this study to analyze
the genetic relationships among Med-
icago sativa, Medicago falcata and
Trigonella foenum-graecum, and to
provide guidance for the classification
of some transitional species.
Materials and Methods
Materials
Plant materials The M. sativa, M.
falcata and T. foenum-graecum plants
planted in the same year were select-
ed as the study objects.
Reagents Taq polymerase, dNTPs
and PCR buffer were purchased from
Shanghai Sangon Biological Engineer-
ing Technology & Services Co., Ltd.
Methods
DNA extraction Firstly, 15 plants
were randomly sampled from each
species, and the DNA of them was
separately extracted using CTAB
method [22]. The DNA quality was de-
tected through 0.8% agarose gel elec-
trophoresis and the DNA concentra-
tion was measured by UV spec-
trophotometer. Then the DNA prod-
ucts were stored at -20℃.
Sample analysis Primers were de-
signed by referring to dinucleotide re-
peat ISSR primers proposed by the
Columbia University, Canada, and
were synthesized by Shanghai San-
gon Biological Engineering Technolo-
gy & Services Co., Ltd.
PCR reaction system (25 μl) con-
tained 1.5 U of Taq DNA polymerase,
2.0 mmol/L of 10 × PCR buffer (with
Mg2+), 0.5 ng/μl of template DNA, 0.6
mmol/L of dNTPs, and 0.9 μmol/L of
each primer.
The PCR was started with prede-
naturing at 94 ℃ for 5 min, followed by
35 cycles of denaturing at 94 ℃ for 30
s, annealing at 55 ℃ for 30 s, and ex-
tension at 72℃ for 30 s; the amplifica-
tion was completed by holding the re-
action mixture at 72 ℃ for 4 min. The
PCR product was detected by elec-
trophoresis on 1.5% agarose gel for 40
min.
Data analysis The PCR products
which were amplified by the same
primers and had the same elec-
trophoretic mobility were considered to
be homologous. The bright and re-
DOI:10.16175/j.cnki.1009-4229.2012.10.021
Agricultural Science & Technology
Vol.13, No.10, 2012 Agricultural Science & Technology
2012
Table 1 Primer sequences and annealing temperatures
Primers Nucleotide sequences Annealing temperatures∥℃
UBC808 AGAGAGAGAGAGAGAG C 55
UBC810 GAGAGAGAGAGAGAGAT 65
UBC807 AGAGAGAGAGAGAGAGT 55
UBC825 CACACACACACACA CAT 55
PRIMER21 AGAGAGAGAGAGAGAG CC 62
UBC853 TCTCTCTCTCTCTCTCRT 55
UBC818 CACACACACACACACAG 53
UBC834 AGAGAGAGAGAGAGAGYT 53
UBC854 TCTCTCTCTCTCTCTCRG 55
UBC862 AGCAGCAGCAGCAGCAGC 61
UBC812 GAGAGAGAGAGAGAGAA 52
UBC866 CTCCTCCTCCTCCTCCTC 54
UBC856 ACACACACACACACACYA 58
UBC836 AGAGAGAGAGAGAGAGYA 56
Table 2 DNA amplification from Medicago sativa, Medicago falcata and Trigonella foenum-
graecum using 14 pairs of primers
Primers Number of polymorphicbands
Total number of
bands
Percentage of
polymorphic bands∥%
UBC808 12 12 100
UBC810 5 5 100
UBC807 4 5 80
UBC825 6 6 100
PRIMER21 4 4 75
UBC853 6 6 100
UBC818 3 3 100
UBC834 6 6 100
UBC854 4 4 100
UBC836 9 9 100
UBC856 10 10 100
UBC862 5 5 100
UBC866 8 8 100
UBC812 9 9 100
Total/Average 91 92 98.57
peatable electrophoretic bands were
calculated using the software POP-
GENE VERSION 1.31[23]. The banding
profiles generated by each primer
were recorded as binary data of “1” or
“0” (presence or absence of bands).
The length of the DNA fragments was
estimated with reference to DL 2000
DNA Marker.
Results and Analysis
Detection of genomic DNA
Agarose gel electrophoresis re-
sults indicated that the genomic DNA
extracted from each study object pro-
duced a clear and complete band,
without dispersion.
Primers screening
In this study, a total of 14 primers
which could produce clear, polymor-
phic and reproducible bands were se-
lected for ISSR test. Their sequences
and an nealing temperatures were
shown in Table 1.
Polymorphism analysis
As shown in Fig.1 and Table 2,
the screened 14 ISSR primers were
used to amplify 18 materials of M. sa-
tiva, M. falcata and T. foenum-grae-
cum plants. A total of 92 bands were
produced and 6.57 bands were pro-
duced by each primer on average.
Primer UBC808 produced the most,
12 bands; and primer UBC818 pro-
duced the lest, only three bands. Am-
ong the 92 bands, 91 were polymor-
phic, accounting for 98.57%. Namely,
6.5 polymorphic bands were produced
by each primer, with the polymor-
phisms ranged from 80% to 100%. As
most primers gave high portion of poly-
morphic information, the ISSR poly-
morphism of the materials was rela-
tively high, indicating that the genetic
basis of the test material was relatively
wide.
Clustering analysis based on ISSR
marker
It could be concluded from Table
3, Table 4 and Fig.2 that similarity co-
efficient among M. sativa, M. falcata
and T. foenum-graecum reached
0.85 -0.94. M. sativa and T. foenum-
graecum shared the smallest similarity
coefficient, 0.85, and the largest ge-
netic distance 4.69, indicating that the
genetic relationship between them
was most distant. The similarity coeffi-
cient betweenM. falcate andT. foenum-
graecum was 0.86. M. falcata and
M . sativa shared the largest similar-
ity coefficient, 0.94 and the smallest
genetic distance , indicating that
T . foenum -graecum shared closer
genetic relationship with M. falcate
rather than M. sativa.
Conclusions and Discus-
sions
ISSR molecular marker technolo-
gy has been widely used in the studies
of plant genetic diversity and phyloge-
netic relationship, and generated reli-
able results[11-22]. But there are too many
factors which could influence the ac-
curacy of ISSR marker, including al-
most all the factors in reaction system
and procedures[24-32]. The errors exist-
ing in ISSR marker inevitably cause
some problems in the analysis of ge-
netic diversity and phylogenetic rela-
M, DL 2000 DNA Marker; 1-5, Trigonella foenum-graecum; 6-11, Medicago sativa; 12-18,
Medicago falcata.
Fig.1 ISSR bands of Medicago sativa, Medicago falcata and Trigonella foenum-graecum
using primer UBC812
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Agricultural Science & Technology Vol.13, No.10, 2012
2012
Fig.2 Clustering analysis about Medicago sativa, Medicago falcata and Trigonella foenum-
graecum based on ISSR marker.
Table 3 Gene similarity among Medicago sativa, Medicago falcata and Trigonella foenum-
graecum
popID
popID
1 2 3
1 1 0.846 1 0.861 2
2 0.167 1 1 0.937 6
3 0.149 4 0.064 5 1
pop1, Trigonella foenum-graecum; pop2,Medicago sativa; pop3,Medicago falcata.
Table 4 Genetic distance among Medicago sativa, Medicago falcata and Trigonella
foenum-graecum
No. Study objects Genetic distance
1 2 and pop1 7.91
2 2 and 1 4.69
3 1 and pop2 3.22
4 1 and pop3 3.22
pop1, Trigonella foenum-graecum; pop2,Medicago sativa; pop3,Medicago falcata.
tionship. In this study, form the 22
primers primarily selected, 14 primers
were finally selected after elec-
trophoresis and used for analysis of
genetic relationship. To some extent,
the primers selection ensured the ac-
curacy of the test, and more accurately
reflected the information in materials
and the genetic relationship among the
test materials.
Because both Medicago and
Trigonella belong to subfamily Legu-
minosae, study on the genetic rela-
tionship among M. sativa , M. fal-
cate and T. foenum-graecum will be
conducive to the classification of those
transitional species. In this study, ge-
netic relationship between Medicago
and Trigonella was investigated and
clarified, which provides reference for
the classification of those transitional
species.
In this study, M. falcata was also
introduced. The result showed that
T. foenum-graecum shared closer ge-
netic relationship with M. falcata than
M. sativa. This result revealed the im-
portance of this study. However, the
problem is that we need a boundary to
identify the transitional species to be
Medicago or Trigonella. So, such a
boundary should be find out in further
researches.
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[19] LIANG HH(梁洪卉), CHENG Z(程舟),
2078
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Vol.13, No.10, 2012 Agricultural Science & Technology
2012
多聚磷酸盐激酶(PPK)基因植物表达载体的构建
曹 访，杨志红 *，韩志萍，杨 倩，费佳玲 （湖州师范学院，浙江湖州 313000）
摘 要 [目的]构建融合表达 PPK和绿色荧光蛋白的融合表达载体 pCAMBIA1302-PPK。[方法]根据 GenBank中登录的大肠杆菌 PPK基因序列
（L03719）设计引物，以 E.coli DH5α基因组 DNA为模板，通过 PCR扩增得 PPK基因，然后用 In-Fusion@ HD Cloning Kit将 PPK基因克隆到
pCAMBIA1302载体的 Nco I酶切位点。[结果]序列测定结果显示，pCAMBIA1302-PPK含有约 2.0 kb的 PPK基因片段，说明 PPK基因已经插入植
物表达载体 pCAMBIA1302的绿色荧光蛋白基因前。[结论]成功构建了融合表达 PPK和绿色荧光蛋白的融合表达载体 pCAMBIA1302-PPK。
关键词 大肠杆菌；多聚磷酸盐激酶基因；植物表达载体；构建
基金项目 国家自然科学基金（31070451）；浙江省钱江人才计划项目（2009R10016）；浙江省自然科学基金（Y5110067）。
作者简介 曹访（1980-），男，江苏丰县人，实验师硕士，主要从事植物转基因技术的研究，E-mail：caofang@hutc.zj.cn*通讯作者，讲师博士，主要从
事植物转基因技术的研究，E-mail：yangzhihong@hutc.zj.cn。
收稿日期 2012-08-28 修回日期 2012-09-14
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
(Continued from page 2075)
利用 ISSR标记解析紫花苜蓿、黄花苜蓿和胡卢巴属植物的亲缘关系
刘 磊 1，2，王宗礼 1，李志勇 1*，周国栋 1，2，师文贵 1，李鸿雁 1，蔡丽艳 1 （1.中国农业科学院草原研究所，内蒙古呼和浩特 010010；2.中国农业科学院
研究生院，北京 100081）
摘 要 [目的]解析紫花苜蓿、黄花苜蓿和胡卢巴属植物的亲缘关系。[方法]利用 ISSR分子标记技术，对紫花苜蓿、黄花苜蓿和胡卢巴种质资源之
间的亲缘关系进行研究。[结果]紫花苜蓿、扁蓿豆和黄花苜蓿有较广遗传基础，胡卢巴种质材料相对于紫花苜蓿种质材料与黄花苜蓿的亲缘关系
更近。苜蓿属和胡卢巴属亲缘关系研究有利于对一些存在争议的中间过渡类型进行分类，但在对中间过渡类型分类时应找一个合适的度，以鉴
定存在争议的中间过渡类型属于胡卢巴属还是苜蓿属。[结论]该研究结果为一些植物材料中间过渡类型的分类学研究提供了指导。
关键词 紫花苜蓿；胡卢巴；黄花苜蓿；亲缘关系；ISSR
基金项目 农业部牧草种质资源保护项目。
作者简介 刘磊（1981-），男，内蒙古乌兰察布人，博士研究生，助理研究员，从事牧草种质资源及牧草育种研究，E-mail:liu4311755@163.com。*通
讯作者，研究员，博士，硕士生导师，从事牧草种质资源研究，E-mail:zhiyongl@public.hh.nm.cn。
收稿日期 2012-06-20 修回日期 2012-08-30

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