全 文 ：作物学报 ACTA AGRONOMICA SINICA 2008, 34(8): 1480−1483 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

Foundation items: Supported by Shanghai Science and Technology Committee (04DZ05003)
Biography: FANG Xue-En (1984–), graduate student, mainly engages in researches about plant molecular biology. E-mail: jackling008@yahoo.com.cn
Received(收稿日期): 2007-12-16; Accepted(接受日期): 2008-03-14.
DOI: 10.3724/SP.J.1006.2008.01480
Application of ISSR in Genetic Relationship Analysis of Sorghum
Species
FANG Xue-En1, CHEN Qin1,*, YIN Li-Ping2, and WANG Wei1
(1 School of Life Science, Shanghai University, Shanghai 200444; 2 Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China)
Abstract: The genetic relationship among six sorghum species, namely, S. bicolor, S. bicolor×S. sudanese, S. saccharatum, S.
sudanese, S. almum, and S. halepense was analyzed by inter-simple sequence repeat (ISSR) method. The results showed that the
diversity of sorghum was high at DNA level. Twenty-two primers selected from 110 ISSR primers could amplify 182 clear and
reproducible bands, of which 153 bands were polymorphic, accounting for 84.0%. All the sorghum species studied could be dis-
tinctly divided into two major groups with the genetic distance level at 0.25 by cluster analysis based on the Neighbor-Joining
method. Some primers produced highly polymorphic band patterns in different species, such as IR 89, IS16, according to them, all
the sorghum species used could be identified. The results could be used in classification, identification and evolution of sorghum.
Keywords: Sorghum; ISSR; Genetic relationship
ISSR在高粱属植物遗传关系研究中的应用
方雪恩1 陈 沁1,* 印丽萍2 王 伟1
(1上海大学生命科学学院, 上海 200444; 2上海出入境检验检疫局, 上海 200135)
摘 要: 以高粱、杂交高粱、甜高粱、苏丹草、黑高粱和假高粱 6种不同高粱属植物为实验材料, 利用 ISSR分子标
记分析其遗传关系。结果表明, 这 6种高粱属植物在 DNA 水平上具有较高的遗传多样性。从 110条 ISSR 引物中成
功筛选到 22 条多态性高、稳定性好的引物, 共扩增出 182 条带, 其中差异性条带 153 条, 多态条带比率(PPB)为
84.0%。基于遗传距离系数的 Neighbor-Joining 聚类分析可以将 6 种高粱分为两大组, 相互之间的遗传距离为 0.25。
另外可以根据一些多态性高的引物所扩增得到的品种特异性条带区分所有不同高粱种, 如引物 IR89、IS16等。这些
为研究高粱属植物的分类、鉴定和进化提供了分子生物学方面的理论依据。
关键词: 高粱属植物; ISSR; 遗传关系
Sorghum is an annual or perennial herbaceous plant
with more than 50 species, possibly possessing considerable
complex genetic relationship, while most of them are eco-
nomic crops which are known for their nutritious value of
grains. However, in recent years, some species have de-
clined rapidly for the reason of being impacted by some
malignant weeds, such as Johnsongrass or Blacksorghum[1-3].
Morphological and enzymatic markers have typically been
used to identify this weeds and study the genetic relationship
of sorghum[4-6], However, the similarities in morphology
make it difficult to identify some species’ seeds. At the same
time, some characters of seeds may be destoryed during
transportation.
ISSR is defined as the amplification of regions be-
tween adjacent microsatellites using a simple sequence re-
peat (SSR) primer. Compared with other molecular markers,
ISSR uses longer primer allowing for higher annealing tem-
perature that results in greater reproducible bands[7]. Be-
cause of the high polymorphism and stability, ISSR have
been employed successfully in analyzing the genetic rela-
tionship of plant species. Chen[8] studied the genetic diver-
sity of 56 individuals of Isoetaceae from China using ISSR;
Song[9] successfully established the polymorphic ISSR pat-
tern in aquatic weed Leersia hexandra. Other plants such as
common wheat[10], melon germless[11], sugarcane[12], Paeo-
nia lactiflora[13], Capsicum frutescens[14], and Paeonia
suffruticosa[15] etc. have already been investigated using
ISSR markers. Nevertheless till now, there was no report
available for the use of ISSR in analyzing sorghum. In the
present study, ISSR technology was used to analyze its
genetic relationship.
1. Materials and methods
1.1 Plant materials
Six representative sorghum species of 30 individuals
第 8期 FANG Xue-En et al.: Application of ISSR in Genetic Relationship Analysis of Sorghum Species 1481

each (Table 1) were all provided by Shanghai Entry-Exit
Inspection and Quarantine Bureau.


Table 1 Sample list
Species Latin name Origin Sample size
Sorghum S. bicolor Afghanistan 30
Hybrid sorghum S. bicolor× S. sudanese USA 30
Sweet sorghum S. saccharatum China 30
Sudangrass S. sudanese USA 30
Blacksorghum S. almum Australia 30
Johnsongrass S. halepense USA 30

1.2 DNA extraction
Total genomic DNA was extracted from frozen young
leaves following the CTAB procedure described by Scott[16].
1.3 ISSR-PCR
One-hundred and ten ISSR primers (Shanghai Sangon)
were used to amplify the genomic DNA. The amplification
was performed in a Thermocycler of FGENO2TD PCR
(TECHNE, America) and commenced with 12 min at 94˚C,
followed by 40 cycles of 30 s at 94˚C, 30 s at 48˚C, 1 min at
72˚C, and ended with 12 min at 72˚C. Reactions were car-
ried out in a volume of 20 μL containing 1×PCR buffer
(TaKaRa, Japan), 2.0 mmol L−1 MgCl2, 250 μmol L−1 dNTP,
400 nmol L−1 primer, 30 ng DNA template, 1.5 U Taq poly-
merase. The amplified products were separated on 1% aga-
rose gel in 1×TAE buffer with the voltage of 80 V for 1h and
visualized by staining with ethidium bromide [17].
1.4 Data analysis
Genetic diversity among different species was meas-
ured by PPB. The cluster analysis was carried out using the
SPSS 10.0 software based on the genetic distance according
to Neighbor-Joining method[18].
PPB (Percentage of polymorphic bands) = Number of
polymorphic bands / Total amplified bands.
2 Results
2.1 Screening of primers and genetic diversity analysis
Of the 110 ISSR primers screened, 22 primers were
selected in our analysis for the clear, reproducible and po-
lymorphic DNA amplification patterns (Table 2). These se-
lected primers generated 182 bands ranging in size from 100
to 2 000 bp, corresponding to an average of 8.27 bands per
primer, and 153 bands were divergent, accounting for 84.0%.
Every primer selected produced polymorphic bands with
PPB value ranging from 63.0% to 100%. Among the 22 se-
lected primers, seven of them gave low PPB values while the
others showed higher polymorphic fingerprints. Some prim-
ers, such as IS31, IR7, IR35 exhibited the highest poly-
morphism with PPB values at 100%. The genetic diversity

Table 2 Sequence of reliable ISSR primers and the number of scorable bands of each prime
Prime
Sequence
(5′–3′)
Total amplified
bands
Number of
polymorphic bands
Percentage of
polymorphic bands
IS13 AGAGAGAGAGAGAGAGA 9 8 89.00
IS15 AGAGAGAGAGAGAGACG 11 9 82.00
IS16 AGAGAGAGAGAGAGACC 14 11 79.00
IS18 AGAGAGAGAGAGAGAC 11 8 73.00
IS23 CAGCAGCAGCAGCAGT 9 8 89.00
IS24 CAGCAGCAGCAGCAGG 9 8 89.00
IS27 CTCTCTCTTCTCTCTG 6 5 83.00
IS31 GCACACACACACACACA 11 11 100.00
IR7 GAGAGAGAGAGAGAGAT 7 7 100.00
IR30 TCTCTCTCTCTCTCTCA 6 5 83.00
IR35 ACACACACACACACACT 7 7 100.00
IR37 TGTGTGTGTGTGTGTGC 8 7 88.00
IR43 AGAGAGAGAGAGAGAGYC 5 5 100.00
IR45 TATATATATATATATART 7 5 71.00
IR46 TATATATATATATATARC 7 6 86.00
IR47 TATATATATATATATARG 7 7 100.00
IR52 CTCTCTCTCTCTCTCTRC 7 7 100.00
IR59 GTGTGTGTGTGTGTGTYG 7 6 86.00
IR74 CTCCTCCTCCTCCTC 8 5 63.00
IR75 GGCGGCGGCGGCGGCGG 9 7 78.00
IR80 CATACATACATACATA 6 4 67.00
IR89 VBVATATATATATATAT 11 7 64.00
Total 182 153 —
Average 8.27 6.95 84.00
1482 作 物 学 报 第 34卷

among the six sorghum species was considerably high ac-
cording to the PPB value of 84%. Otherwise, the results
showed that the di-nucleotide repeat primers could result in
the ideal band patterns while among 22 primers selected,
only four tri-nucleotide repeat primers (IS23, IS24, IR74,
IR75) and one tetra-nucleotide repeat primer (IR80) could
have clear patterns, which accorded well with the fact that
targeted unit di-nucleotides are more abundant in sorghum
species.
2.2 Cluster analysis
Based on the Neighbor-Joining method, a cluster
analysis was carried out and a dendrogram was generated
that represented the genetic relationships among six sor-
ghum species (Fig. 1). In the dendrogram, all the sorghum
species were distinctly separated into two major groups,
group I (Blacksorghum and Johnsongrass) and group II
(sweet sorghum, sudangrass, sorghum and hybrid sorghum)
at the genetic distance level of 0.25. The genetic distance
level in group I was 0.21, much higher than group II; sweet
sorghum and Sudangrass could be clustered together while
sorghum and hybrid sorghum into another in group II with
the genetic distance level ranging from 0.075 to 0.180.



Fig. 1 Dendrogram illustrating genetic relationship among six
sorghum species
2.3 Identification of sorghum species
According to the patterns obtained with 22 selected
primers, all the sorghum species analyzed could be distin-
guished, especially the primers IR89 and IS16, as shown in
Fig. 2 and Table 3. According to the specific bands in 1 500
bp and 100 bp, Blacksorghum and Johnsongrass could be
separated from the other four sorghum species (Fig. 2, left).
According to Fig. 2 (right), the Black sorghum had a spe-
cies-specific band in 400 bp, which could be separated from
the Johnsongrass; the hybrid sorghum had a distinctly spe-
cific band in 850 bp which could be used to identify it.
Meanwhile, Sudangrass was lack of the band in 1 200 bp
which could be used to distinguish it. In a word, the different
sorghum species could all be well identified by the two
primers. Other primers, such as IR30, IR35, IS13, and IS27
shown in Table 4 obtained relatively few bands, but acquired
some species-specific bands, which could be used as mo-
lecular makers to identify some sorghum species. The other
primers resulted in complex bands, which can assist to dis-
criminate with each other. Above all, it was anticipated that
ISSR-PCR could be exploited as the basis of molecular
techniques for sorghum species identification.



Fig. 2 Amplification of genome in six sorghum species using
primer IR89 (left) and IS16 (right)

Table 3 Specific bands of sorghum by IR89 and IS16
Prime Fragment length (bp) Sorghum Hybrid sorghum Sweet sorghum Sudangrass Blacksorghum Johnsongrass
IR 89 1500 − − − − + +
100 + + + + − −
IS16 1200 + + + − − −
1100 + + − − − −
850 − + − − − −
400 + + + + + −
‘‘+’’: specific band; ‘‘–’’: no relevant band.

Table 4 Species-specific bands from different primer amplifications
Primer Fragment length (bp) Sorghum Hybrid sorghum Sweet sorghum Sudangrass Blacksorghum Johnsongrass
IS27 1200 + − − − − −
IS31 1200 + − − − − −
IR35 1800 − + − − − −
IR37 1100 − + − − − −
IR75 250 − − − + − −
IS13 1500 − − − − + −
IS24 1750 − − − − + −
IR30 2000 − − − − + −
IR46 1700 − − − − − +
IR31 250 − − − − − −
IR43 2200 − − − − − −
IR45 1500 − − − − − −
‘‘+’’: specific band; ‘‘–’’: no relevant band.
第 8期 FANG Xue-En et al.: Application of ISSR in Genetic Relationship Analysis of Sorghum Species 1483

3 Discussion

To the best of our knowledge, this is the first report of
genetic diversity in sorghum detected by ISSR primers. The
ISSR analysis of six sorghum species revealed a high level of
genetic diversity with the PPB value at 84.0% — much higher
than detected by morphological traits, allozymes, or microsatel-
lites [4-6]. This phenomenon in sorghum may be interpreted as
occasional sexual recruitments in some habitats or the geo-
graphic isolation between populations. The diversity level be-
tween the different sorghum species revealed by ISSR has great
significances in sorghum species conservation and breed-
ing[19-20]. In the study, di-nucleotide repeat primers are more
suitable to be used in analyzing the genetic relationship of sor-
ghum than tri-, tetra-, and penta-nucleotide repeat primers,
which consist well with the knowledge that targeted unit
di-nucleotides are more abundant in sorghum [21]. Meanwhile,
poly (AG)-anchored ISSR primers produced more bands in
sorghum species than the other di-nucleotide repeat primers
which suggests that the frequency of poly (AG) in the sorghum
species genome is higher than that of poly (CT), poly (AT),
poly (CG), poly (AC), and poly (GT).
Cluster analysis was carried out based on the ISSR data
by SPSS 10.0 software, which showed that all sorghum species
studied could be clustered into two groups. Group I was com-
posed of Johnsongrass and Blacksorghum, which accorded well
with the fact that the Blacksorghum is the hybrid between
Johnsongrass and an unknown sorghum species [22]. Interest-
ingly, this two sorghum species are all malignant weeds which
will cause serious reduction to crops and bring serious financial
loss once invading into an area; Group II was composed of
sorghum, hybrid sorghum, sweet sorghum and Sudangrass, all
of them are known for its ability to survive under the severe
environmental conditions and for the nutritious value of their
grains. They are the major economic crops in the world.
ISSR technique was further more applied to identify dif-
ferent sorghum species. According to the patterns obtained with
some high polymorphic primers, all sorghum species studied
could be successfully discriminated which has important impli-
cations for preventing the malignant weeds, such as Blacksor-
ghum and Johnsongrass. Such a method will be a useful identi-
fication tool only if a reference sample from the sorghum spe-
cies is present in the database. In this study, only six sorghum
species were analyzed, further investigations will include the
analysis of more species to allow the resolution of species
status more detailed and to establish a reliable, quick and con-
venient authentication system for sorghum species [22]. In a
word, with the advantages of high polymorphism, reproducibil-
ity and convenience, ISSR could offer a quick and reliable al-
ternation in analyzing the genetic relationship of sorghum and
identification of some malignant species.
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