ISSN 10227954, Russian Journal of Genetics, 2012, Vol. 48, No. 6, pp. 663–666. © Pleiades Publishing, Inc., 2012.

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Identification of a Specific SCAR Marker for Detection

of Tilletia foetida (Wall) Liro Pathogen of Wheat1
M. Zhanga,*, W. Q. Chenb, *, D. Liua, T. G. Liub, L. Gaob, and K. Shua, c
a
Department of Plant Pathology, Sichuan Agricultural University, Ya’an, Sichuan 625014, P.R. China
email: yalanmin@126.com
b State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy
of Agricultural Sciences, Beijing 100193, P.R. China
email: wqchen@ippcaas.cn
c
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing 100101, P.R. China
Received May 23, 2011; in final form, September 23, 2011

Abstract—Common bunt is one of the most important destructive diseases of wheat worldwide and is a
domestic quarantined disease in China. However, a rapid and efficient method to identify the corresponding
pathogens is currently limited. The objective of the present study was to develop a diagnostic molecular
marker specific towards Tilletia foetida (Wall) Liro, a causal agent of the bunt disease. One specific DNA frag
ment for T. foetida (286 bp in length) was amplified using an Amplified Fragment Length Polymorphism
(AFLP) assay and, this fragment was cloned and sequenced. One pair of specific primers (SC2861/SC2862),
which was designed according to the sequence, could specifically amplify the corresponding fragment in all
of the T. foetida isolates employed from both the People’s Republic of China and United States, whereas this
fragment could not be amplified by the other fungal species tested. Therefore, a specific Sequence Charac
terized Amplified Region (SCAR) marker was developed. This SCAR marker could distinguish T. foetida
from related pathogenic fungi efficiently and could be used for the early diagnosis of the common bunt of
wheat in the field, and provide an efficient way for disease surveillance and disease forecasting in cereal crop.
DOI: 10.1134/S1022795412050237

*1 Wheat (Triticum aestivum L.) is one of the most
important crops in the world, and also the main food
stuff in China. Common bunt of wheat is caused by
Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.]
and T. laevis Kühn [syn. T. foetida (Wallr) Liro] and
also is one of the most destructive diseases in wheat
[1], ranked second only to the rusts [2]. It occurs on
both spring and winterplanted wheat, and is present
throughout the wheat growing regions worldwide and
has a local distribution in the north of China. In the
diseased head, grains are replaced with fishy smelling,
black bunt balls, and the pathogens are effectively dis
seminated on host seeds. There are no effective
organic compounds for seed treatment approved in the
field, so there is a greater probability of high rates of
infection in organic production [3]. A rapid and effi
cient strategy for identification of T. foetida is cur
rently limited. Therefore, an efficient method of
detection for the corresponding pathogens is essential
to forecast the disease.
Many traditional methods for detection of the
pathogens were mostly covered based on microscopic
examination of the teliospores [4], immunological
1 The article is published in the original.
* These authors contributed equally to this work.
methods [5], analyses of polypeptide profiles [6], tria
cylglycerol profiles [7], and genetic properties of the
teliospores [8]. All of these methods have limitations
including being time consuming or having a low accu
racy, so that they could not satisfy the requirements of
rapid and accurate diagnosis. It is imperative to
develop rapid and accurate methods for detecting
these pathogens.
Molecular diagnosis has been widely used in ani
mal, plant, microbe, and humans [9]. The Amplified
Fragment Length Polymorphism (AFLP) technique,
based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA, is an
efficient molecular technique for DNA research, and
also is a powerful DNA fingerprinting technique [10].
It also has been advocated as powerful tool for species
identification during recent decade [11]. However,
since T. foetida possess as close genetic compatibility
to many related pathogens, such as T. controversa,
T. caries, and frequent genetic recombination leads to
a great deal of variability among these fungal species,
AFLP analysis is ineffective to differentiate among
them accurately. In addition, AFLP markers are not
suitable for largescale detection of pathogens [12,
13]. In contrast, Sequence Characterized Amplified
Region (SCAR) markers, based on AFLP or Random

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1 2 3 4 5 6 7 8 9 10 11
286 bp
Fig. 1. Amplified fragment length polymorphism pattern
on gel and the specific fragment (arrow indicated) for
T. foetida obtained by primer combination M09/E04.
Lanes 1–4, Tilletia foetida (Wall) Liro; 5, negative control;
6, T. controversa Kuhn; 7–9, T. caries (DC.) TuI.; 10,
Sphaceletheca reiliana (Kuhn) Cling; 11, Ustilago tritici
(Pres.) Rostr.
Amplified Polymorphic DNA (RAPD) panalysis have
been used to produce specific and reproducible results
for detection of pathogens [14]. More recently, reliable
and specific SCAR marker or PCRbased diagnostic
strategies have been successfully constructed for Tille
tia caries [15] and Tilletia controversa [16, 17].
The shortage of the available specific molecular
markers remains a barrier for rapid detection of T. foe
tida. Our objective was to develop a fast, robust SCAR
marker for detection of this pathogen. An accurate
marker could be used for the early diagnosis of com
mon bunt of wheat in the field, and provide an effi
cient way for surveillance and forecasting of disease in
cereal crops.
Five isolates of T. foetida for test were collected
from Yiyang and Fangcheng in Henan Province,
Gonghe in Qinghai Province, Yili in Xingjiang
Municipality of P. R. China, and Washington State of
the USA, respectively. Other related species including
one isolates of T. caries, three isolate of Ustilago tritici
(Pres.) Rostr., and one isolate of Sphaceletheca reili
ana (Kuhn) Clint, were obtained from Henan, Gansu,
Beijing, Sichuan and Xingjiang provinces (or Munici
pality) of P.R. China, separately, and one isolate of
T. controversa was supplied kindly by Dr. Blair Goates
in National Small Grains Germplasm Research Facil
ity, USDAARS. All of fungi isolates used in experi
ment were identified by and conserved in our labora
tories. The different isolates of T. controversa,
T. caries, U. tritici and S. reiliana were used as con
trols.
The fungal strains used in these experiments were
conserved in 15% glycerol at –70°C as previously
described by Loomins and Leung [18]. The different
fungus isolates were cultivated according to the meth
ods outlined by Shi et al. [13]. The DNAs of the differ
ent fungal isolates were extracted according to the
ZHANG et al.
assay of Graham et al. [19]. AFLP analysis protocols
were adapted from Vos et al. [10] minor modifications.
Approximately 500 ng DNA of each sample was
digested by EcoRI/MseI complex restriction enzyme
solution, which contained 5 U EcoRI and MseI, 8 μL
10× Buffer Tango™, 26 μL ddH2O, and the proce
dures following were at 37°C for 2 h for EcoRI, at 65°C
for 2 h for MseI, and then at 85°C for 30 min for ter
mination. Consequently, 5 pMol EcoRIadapters,
50 pMol MseIadapters, 1 U T4 DNAligase, 1 mMol
ATP, 2 μL 10× Buffer TangoTM, and 4.9 μL ddH2O
were added into the former solutions in order, and
incubated at 22°C for 10 h, and then at 65°C for
15 min. The ligation product was preamplified in
30 μL of PCR reaction mixture, using MJ
RESEARCH (PTC200) thermocycler, EcoRI and
MseI primers without any additional selective nucle
otide at the 3' end, and amplification conditions were
30 cycles at 94°C for 30 s, 56°C for 60 s, and 72°C for
60 s. After preamlification, the template was diluted in
water 1 : 30 for selective amplification. Randomly
selected primer pairs were employed for selective
amplification. The PCR products were loaded on 8 M
Urea and 1× TBE [90 mM Trisborate (PH 8.3), 6%
polyacrylamide (19 : 1 acrylamide : Bis), 2 mM
EDTA] gels, which were run at 80 W for approxi
mately 1.5 h and visualized using silver staining
according to the method described elsewhere [20].
The specific fragment was detected after silver
nitrate staining and, the gel containing the critical
fragment was excised. The fragment was eluted with
25 μL 1× TE buffer. Consequently, 1 μL of the solution
was used as template for polymerase chain reaction
(PCR) amplification with the same primer pair. The
specific DNA band was identified again by silver
nitrate staining of a gel and, followed by the cloning
and sequencing of the fragment.
The identified specific DNA fragment was excised
from the gel and purified, then ligated into the
PGEMT easy vector according to the procedures
outlined by the manufacturer [Sino Geno Max Co.,
Ltd (Beijing)]. The transformation of host strain
Escherichia coli JM109 under the competent cells was
executed. The recombinant plasmids were screened
on the selective LuriaBertani media containing
ampicillin (50 ug mL–1). To check the insertion of the
critical fragment, DNA of the plasmids was extracted
and the DNA was digested by EcoRI/MseI followed by
PCR amplification. Consequently, the corresponding
purified plasmid DNAs which amplified a single spe
cific amplification product were sequenced by Sino
Geno Max Co. Ltd (Beijing). One primer pair was
designed by Primer Premier 5.0, as a specific primer
according to the sequence of the specific fragment.
This SCAR marker, namely SC2861/SC2862, was used
to screen all the fungus isolates including T. foetida,
T. controversa, T. caries, U. tritici and S. reiliana by
PCR to validate it.
RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 6 2012