Mol Biotechnol (2012) 50:5761

DOI 10.1007/s12033-011-9415-5

RESEARCH

Development of SCAR Marker Specific to Non-Toxic Jatropha

curcas L. and Designing a Novel Multiplexing PCR Along
with nrDNA ITS Primers to Circumvent the False Negative
Detection
Shaik G. Mastan Pamidimarri D. V. N. Sudheer
Hifzur Rahman Muppala P. Reddy
Jitendra Chikara

Published online: 10 May 2011

Springer Science+Business Media, LLC 2011

Abstract Jatropha curcas L., a multipurpose shrub, has

acquired significant economic importance for its seed oil
which can be converted to biodiesel an emerging alternative to petro-diesel. In addition to the commercial value, it
is also having medicinal and even high nutritional value to
use as animal fodder which is limited due to the toxicity.
Development of molecular marker will enable to differentiate non-toxic from toxic variety of J. curcas in a mixed
population and also for quality control since the toxic
components of J. curcas has deleterious effect on animals.
In the present study, the efforts were made to generate the
specific SCAR marker for toxic and/or non-toxic J. curcas
from RAPD markers. Among the markers specific for toxic
and non-toxic varieties, four were selected, purified,
cloned, sequenced, and designed primers out of which one
set of primers NT-JC/SCAR I/OPQ15-F and R could able
to discriminate the non-toxic with toxic Jatropha by giving
expected 430 bp size amplification in non-toxic variety.
Furthermore, novel multiplex PCR was designed using the
nrDNA ITS primers to overcome the false negatives.
Present work also demonstrates utility of the conserved
regions of nrDNA coding genes in ruling out the artifacts in
S. G. Mastan  H. Rahman  J. Chikara
Discipline of Wasteland Research, Central Salt and Marine,
Chemicals Research Institute (CSIR), Bhavnagar,
Gujarat 364002, India
P. D. V. N. Sudheer (&)
Guru Ghasidas Vishwavidyala (A Central University), Koni,
Bilaspur 495009, Chhattisgarh, India
e-mail: Sudheer.vsc@gmail.com
M. P. Reddy
Plant Stress Genomics and Technology Center, King Abdullah
University of Science and Technology, Thuwal 23955-6900,
Kingdom of Saudi Arabia

PCR-like false negatives frequently occur in SCAR due to

various reasons. The specific SCAR markers generated in
the present investigation will help to distinguish non-toxic
from toxic varieties of J. curcas or vice versa, and isolated
marker along with designed multiplex protocol has applications in quality control for selective cultivation of nontoxic variety and will also assist in breeding and molecular
mapping studies.
Keywords Biofuel  Multiplex PCR  Jatropha curcas 
SCAR  Non-toxic genotype

Introduction
Jatropha curcas L., belonging to the family Euphorbeaceae, is native to South America and widely distributed in
South and Central America, Africa, and Asia. J. curcas is a
multipurpose shrub with significant economic importance
and has ability to rehabilitate the degraded lands [1]. Since
its seed oil can be converted to biodiesel, it is emerging as a
renewable energy source, alternative to petro-diesel and is
highly promoted for large scale cultivation and production
of biodiesel. Several reports demonstrated better performance of the Jatropha biodiesel over conventional petrodiesel [13]. In spite of high-nutritional composition, seed
cake obtained from the toxic J. curcas remains unutilized
as animal feed due to its toxic nature [4, 5], and no successful attempts have been made till now for completely
eliminating toxic principle [6]. Globally, J. curcas is promoted for large acreage cultivation for biodiesel production
[7, 8]. Selective cultivation of non-toxic variety reported
from Mexico, whose innocuous nature was established
[4, 9, 10], will add value to the crop through utilization of
de-oiled seed cake as a safe animal feed.

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Cultivation of non-toxic variety of J. curcas will provide

oil for biodiesel as energy source and de-oiled seed cake as
a live stock feed [5]. No significant morphological, qualitative, and quantitative differences are known between toxic
and non-toxic varieties except for the phorbol ester content
in the toxic variety which makes it difficult to discriminate
these two varieties [1, 4] Development of any simple marker which enables identification of non-toxic variety from
toxic variety will not only add to the quality control for
selective cultivation of non-toxic variety and also avoid any
toxic adulteration in the animal feeds and even in medicinal
use. In our previous study, we found specific makers both
for toxic and non-toxic varieties using RAPD, AFLP, and
SSR marker systems [11]. Generation of SCAR markers
specific for toxic and non-toxic will be an added advantage
in identification and screening of the large number of
samples in short span of time than remaining marker systems. The PCR artifacts due to the template quality and
other practical procedure prone several times to false negatives (absence of amplification) which in turn effect the
identification and quality control. Thus, in the present study,
efforts were made to isolate the SCAR from the specific
markers identified in our previous study to discriminate
toxic with non-toxic variety and also to design a novel
multiplex PCR to preclude the false negatives to make the
protocol easy to identify the non-toxic J. curcas.

Materials and Methods

Genomic DNA Extraction
Genomic DNA was extracted using CTAB protocol as
described by Sudheer et al. [12] from different germplasm
of toxic variety and non-toxic variety of J. curcas established in Central Salt and Marine Chemicals Research
Institute, Bhavnagar, Gujarat, India, experimental field
(21750 N, 72140 E). 0.1 g of leaf tissue was ground in
liquid nitrogen and taken into a 2-ml microcentrifuge tube.
To the ground sample, 0.5 ml of extraction buffer (2%
CTAB, 100 mM TrisHCl, 3.5 M NaCl, 20 mM EDTA,
0.2 M b-Mercaptoethanol, 2% PVP, pH 8.0) was added
and incubated at 65C for 90 min. The above sample was
extracted with equal volume of chloroform: isoamyl alcohol (24:1), and supernatant was transferred into a new tube.
The obtained supernatant was precipitated with 80% of
ethanol. The pellet was air dried and dissolved in 100 ll of
Millipore water or TE.

Mol Biotechnol (2012) 50:5761

(Operon technologies Inc, USA; IDT, USA). The reaction

was carried out in a volume of 25 ll of reaction mixture
containing final concentration of 10 mM TrisHCl, 50 mM
KCl, 0.1 Triton X-100(pH 9.0), 0.2 mM each dNTPs,
3.0 mM MgCl2, 0.4 lM primer, 25 ng template, 1 unit Taq
DNA polymerase (Sigma, USA). Amplification was
performed with different germplasm of toxic variety of
J. curcas and non-toxic germplasm of J. cucas in programmed thermal cycler (Master Cycler EP gradient S,
Eppendorf, Germany) with program of initial denaturation
at 94C for 3 min, 42 cycles of denaturation at 94C for
30 s, primer annealing at 32C for 1 min, extension at
72C for 2.5 min, and final extension at 72C for 4 min.
Amplification products were electrophoresed in 1.5%
agarose in TBE (90 mM Trisborate, 2 mM EDTA, pH 8).
The gels were stained with ethidium bromide and documented using gel documentation system (Syngene, UK).
Experiment was repeated three times with each primer, and
those resulted in reproducible fingerprints were considered
for further marker purification and cloning.
SCAR Marker Isolation
For the SCAR marker isolation, polymorphic RAPD bands
specific to non-toxic Mexican genotype were excised from
the RAPD agarose gel and purified using QIAquick gel
extraction kit (Qiagen, Germany). The purified fragments
were ligated into pTZ57R (Insta) T/A cloning vector (MBI
Fermentas, USA) according to manufacturers protocol,
and then the recombinant plasmids were transformed into
competent E. coli strain (DH5a) by heat shock method
according to Sambrook et al. [14]. The cells were then
spread on LuriaBertani (LB) selection medium containing
appropriate ampicillin, IPTG, and X-gal and incubated at
37C for 16 h. The putative clone selection was carried out
based on blue/white selection and checked for insert by
colony PCR using M13 universal primers. The recombinant plasmids were extracted using GenElute Plasmid
Miniprep Kit (Sigma, USA). The sequences were obtained
using ABI PRISM 3100 Genetic Analyser (Applied Biosystems) with M13 universal primers (IDT, USA). With the
obtained sequence, the forward and reverse primers were
designed including the decamer primer sequence extending
few nucleotides (810 bases). The Tm, GC, and other
factors were analyzed using NetPrimers software and the
final compatible forward and reverse primers selected for
specific SCAR amplification.

RAPD Analysis

Multiplex PCR Reaction for Identification

of Non-Toxic J. curcas

Amplification of RAPD fragments was performed according to Williams et al. [13] using decamer arbitrary primers

For the multiplex PCR reaction, the primers designed from

the sequenced non-toxic specific markers NT-JC/SCARI/

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Mol Biotechnol (2012) 50:5761

OPQ15-F (50 GGGTAACGTGGTGGGGGA30 ) and NT-JC/

SCAR I/OPQ15-R (50 -GGGTAACGTGTAAAAGTATT30 ) and the JCITS-1-F (50 -ACCTGCGGAA GGATCATT
GTCGAAA-30 ) and JCITS-2-R (50 -CCTGGGGTCGC
GATGTGAGCGT-30 ) designed in our earlier studies were
taken for the multiplex PCR in the present study. A total of
50 ng of genomic DNA was used as template in a 15-ll
PCR reaction mixture containing final concentration of
10 mM TrisHCl, 50 mM KCl, 0.1% Triton X-100(pH
9.0), 0.2 mM each dNTPs, 3.0 mM MgCl2, 0.4 lM primer,
and 1 unit Taq DNA polymerase. Amplification was conducted using a thermal cycler (Eppendorf EP Gradient S,
Germany) with the following program: initial denaturation
at 94C for 4 min, 35 cycles at 94C for 30 s, primer
annealing at 55C for 1 min, extension at 72C for 1 min,
and final extension at 72C for 4 min. The amplification
products were separated on 1.5% agarose gel electrophoresis in 19 TAE buffer (0.04 M Trisacetate, 1 mM
EDTA, pH 8) at 80 V for 45 min and subsequently stained
with 0.5 lg/ml ethidium bromide solution for 10 min. The
gel was destained in sterile distilled water (15 min) and
photographed by gel documentation system (Syngene,
USA).

Results and Discussion

Jatropha curcas, a multipurpose shrub, has acquired high
agro-industrial significance globally because of its seed oil
which is a potential source of biodiesel and also for its
beneficial by-products [3, 1518]. Based on the concentration of phorbol esters, J. curcas was categorized into
toxic and non-toxic varieties. These phorbol esters found in
J. curcas were found to be main toxic compound for animals and humans [5, 19, 20]. The seed cake after oil
extraction is toxic as high concentration of phorbol esters
are present in seeds of toxic variety; oil and deoiled cake
are not suitable for animal consumption [5] and cannot be
used as feed despite having high protein content and
favorable amino acid profile. The non-toxic J. curcas has
been reported from Mexico, according to Makker et al. [19]
found to have very low amount of phorbolesters whose
seed cake innocuous nature was established [4, 9, 10]. As
there were no qualitative and quantitative differences were
reported between toxic and non-toxic varieties, development of SCAR marker specific to non-toxic variety will
enable to screen it from mixed populations which aid to
overcome the problem of adulteration. In our previous
studies, we have identified polymorphic markers specific to
both toxic and non-toxic J. curcas successfully using
RAPD, AFLP, and SSR markers [11]. Thus, deriving the
SCAR markers from the identified markers will have better
application and exhibits several advantages over direct

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multilocus markers like (a) Stringent PCR conditions can

be applied that exclude competition between primer binding sites. This results in reliable and reproducible bands
that are less sensitive to reaction conditions; (b) SCAR
markers are locus-specific and are more informative for
genetic mapping than dominant RAPDs or AFLPs. (c) The
reproducible amplification of defined genomic regions
allows comparative mapping and synteny studies between
related species and varieties. Having so many advantages,
the SCAR isolation is laborious and depends on the chance
of amplifying specifically with the designed primers.
However, its high importance in identification in very
stringent conditions made it very superior marker and
many successful attempts leads to many consortium of
SCAR markers for identification of different species,
varieties, cultivars, and populations including the different
populations of J. curcas. In the present study, the efforts
were made to generate the specific SCAR marker for toxic
and non-toxic J. curcas.
SCAR Isolation and Amplification
In our previous study, totally 180 RAPD primers were
screened and 52 primers those resulted more than 6 scorable bands were selected for the analysis. Out of the
primers screened, 39 primers resulted in total 66 polymorphic markers either specific to toxic or non-toxic
J. curcas. Among the identified markers, the efforts were
made to clone the specific DNA bands eluted from the
RAPD agarose gels. Four markers among them were successfully cloned, confirmed by colony PCR (Fig. 1) and
sequenced. The primers were designed to the 50 and 30
regions for the obtained sequence. Among the four sets of
the primers, three sets gave the amplification in both the
varieties. Only one marker isolated from the profiles
of agarose gel sample amplified with primer OPQ15
(50 GGGTAACGTG 30 ) (Fig. 2) has amplified with expected
size of 430 bp selectively in non-toxic Jatropha but not in
toxic (Fig. 3). The experiment was repeated thrice to confirm
the results and to check the consistency of it. We have also
designed multiplex PCR to check the false negative amplification generally occur due to PCR artifacts.
Multiplex PCR
A multiple PCR was designed by taking the conserved
regions of nrDNA ITS region. The primers designed
(JCITS-1-F and JCITS-2-R) and used in our earlier studies
[21] for the phylogenetic analysis of Jatropha species were
used in combination with NT-JC/SCARI/OPQ15 primers
sets (Fig. 4). The nr DNA ITS primers are in the conserved
regions and have the ability to amplify across the species
and gives definite amplification in both toxic and non-toxic

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Mol Biotechnol (2012) 50:5761

Fig. 1 a RAPD profile with

primer OPQ15, 16 different
germplasm of toxic variety and
7 non-toxic variety of J. curcas,
M 1 kb Marker (BioGene, USA)
OPQ15; b 1 purified DNA
fragment of non-toxic-specific
DNA band (NT-JC/SCAR
I/OPQ15) obtained with
OPQ15, M 1 kb Marker;
c confirmation of clone with
insert NT-JC/SCARI/OPQ15,
M 1 kb Marker

Fig. 2 Nucleotide sequence of RAPD amplicon specific to non-toxic J. curcas (NT-JC/SCAR I/OPQ15); arrows represent forward primer
(NT-JC/SCAR I/OPL15-F) and reverse primer NT-JC/SCAR I/OPQ15-R), the underlined sequence belongs to the oligo decamer primer OPQ15

Fig. 3 Amplification with NT-JC/SCARI/OPQ15 primer set in nontoxic variety of J. curcas; lanes 14 non-toxic variety J. curcas; lane
5 toxic variety of J. curcas; lane 6 100 bp Marker

varieties which indicates the no PCR artifacts and acts as

the positive control amplification in all the reaction samples. The Tm for the multiplex without miss amplification
was obtained with gradient PCR and is found to be 50.5C
and consistency of the multiplex PCR was found to be
100% in identification of the non-toxic J. curcas. The
present work demonstrate the high utility of the conserved
regions of the nrDNA coding genes in ruling out the artifacts in PCR-like false negatives frequently occur in SCAR
due to various reasons especially arise because of template

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Fig. 4 Multiplex PCR amplification among different germplasm of

toxic variety and non-toxic variety of J. curcas using primers NT-JC/
SCARI/OPQ15-F, NT-JC/SCARI/OPQ15-R, JCITS-1-F, and JCITS2-R; lane 1 100 bp marker; lanes 2 and 3 amplification by non-toxic
varieties; lanes 49 amplification by toxic J. curcas varieties

quality. The present investigation reports on isolation of the

molecular marker for the identification of non-toxic
J. curcas and application of the multiplex to devoid the
false positives and/or negatives frequently occur in SCAR
analysis. The specific SCAR markers generated will help to
distinguish non-toxic from toxic varieties of J. curcas or
vice versa and isolated marker which specifically identifies
the non-toxic variety along with designed multiplex protocol has huge application in quality control for selective

Mol Biotechnol (2012) 50:5761

cultivation of non-toxic variety, and also avoid any toxic

adulteration in the animal feeds and in breeding and
molecular mapping studies.
Acknowledgments We are thankful to Council for Scientific and
Industrial Research, New Delhi, India, for its financial support and for
research associate fellowship. We would like to thank Professor
B. N. Tiwari, Head, School of biotechnology, for his kind support and
encouragement.

61

10.

11.

12.

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