RESEARCH

Development of SCAR Marker Specific to Non-Toxic Jatrophacurcas L. and Designing a Novel Multiplexing PCR Alongwith nrDNA ITS Primers to Circumvent the False NegativeDetection

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 alterna-

tive 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 differ-

entiate 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

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 appli-

cations in quality control for selective cultivation of non-

toxic 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 Euphorbea-

ceae, 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 perfor-

mance of the Jatropha biodiesel over conventional petro-

diesel [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 suc-

cessful attempts have been made till now for completely

eliminating toxic principle [6]. Globally, J. curcas is pro-

moted 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.

S. G. Mastan H. Rahman J. ChikaraDiscipline 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

123

Mol Biotechnol (2012) 50:5761

DOI 10.1007/s12033-011-9415-5

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, quali-

tative, 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 mar-

ker 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 sys-

tems. The PCR artifacts due to the template quality and

other practical procedure prone several times to false neg-

atives (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 estab-

lished in Central Salt and Marine Chemicals Research

Institute, Bhavnagar, Gujarat, India, experimental field

(21750N, 72140E). 0.1 g of leaf tissue was ground inliquid 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 addedand incubated at 65C for 90 min. The above sample wasextracted with equal volume of chloroform: isoamyl alco-

hol (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 ofMillipore water or TE.

RAPD Analysis

Amplification of RAPD fragments was performed accord-

ing to Williams et al. [13] using decamer arbitrary primers

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

was carried out in a volume of 25 ll of reaction mixturecontaining 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 TaqDNA polymerase (Sigma, USA). Amplification was

performed with different germplasm of toxic variety of

J. curcas and non-toxic germplasm of J. cucas in pro-

grammed 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 for30 s, primer annealing at 32C for 1 min, extension at72C 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 docu-

mented 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 methodaccording 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 outbased on blue/white selection and checked for insert by

colony PCR using M13 universal primers. The recombi-

nant plasmids were extracted using GenElute Plasmid

Miniprep Kit (Sigma, USA). The sequences were obtained

using ABI PRISM 3100 Genetic Analyser (Applied Bio-

systems) 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.

Multiplex PCR Reaction for Identification

of Non-Toxic J. curcas

For the multiplex PCR reaction, the primers designed from

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

58 Mol Biotechnol (2012) 50:5761

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OPQ15-F (50GGGTAACGTGGTGGGGGA30) and NT-JC/SCAR I/OPQ15-R (50-GGGTAACGTGTAAAAGTATT-30) and the JCITS-1-F (50-ACCTGCGGAA GGATCATTGTCGAAA-30) and JCITS-2-R (50-CCTGGGGTCGCGATGTGAGCGT-30) designed in our earlier studies weretaken for the multiplex PCR in the present study. A total of

50 ng of genomic DNA was used as template in a 15-llPCR 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 con-

ducted 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, primerannealing at 55C for 1 min, extension at 72C for 1 min,and final extension at 72C for 4 min. The amplificationproducts were separated on 1.5% agarose gel electropho-

resis 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. Thegel 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 concen-

tration 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 ani-

mals 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, develop-

ment 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

multilocus markers like (a) Stringent PCR conditions can

be applied that exclude competition between primer bind-

ing 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 scor-

able bands were selected for the analysis. Out of the

primers screened, 39 primers resulted in total 66 poly-

morphic 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 suc-

cessfully 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 expectedsize 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 ampli-

fication 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

Mol Biotechnol (2012) 50:5761 59

123

varieties which indicates the no PCR artifacts and acts as

the positive control amplification in all the reaction sam-

ples. The Tm for the multiplex without miss amplification

was obtained with gradient PCR and is found to be 50.5Cand 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 arti-

facts in PCR-like false negatives frequently occur in SCAR

due to various reasons especially arise because of template

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 pro-

tocol has huge application in quality control for selective

Fig. 1 a RAPD profile withprimer OPQ15, 16 differentgermplasm of toxic variety and

7 non-toxic variety of J. curcas,M 1 kb Marker (BioGene, USA)OPQ15; b 1 purified DNAfragment of non-toxic-specific

DNA band (NT-JC/SCAR

I/OPQ15) obtained with

OPQ15, M 1 kb Marker;c confirmation of clone withinsert 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 non-toxic variety of J. curcas; lanes 14 non-toxic variety J. curcas; lane5 toxic variety of J. curcas; lane 6 100 bp Marker

Fig. 4 Multiplex PCR amplification among different germplasm oftoxic variety and non-toxic variety of J. curcas using primers NT-JC/SCARI/OPQ15-F, NT-JC/SCARI/OPQ15-R, JCITS-1-F, and JCITS-

2-R; lane 1 100 bp marker; lanes 2 and 3 amplification by non-toxicvarieties; lanes 49 amplification by toxic J. curcas varieties

60 Mol Biotechnol (2012) 50:5761

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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 andIndustrial 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.

References

1. Ghosh, A., Chaudhary, D. R., Reddy, M. P., Rao, S. N., Chikara,

J., Pandya, J. B., et al. (2007). Prospects for Jatropha methyl ester(biodiesel) in India. International Journal of EnvironmentalStudies, 64, 659674.

2. Takeda, Y. (1982). Development study on Jatropha curcas(Sabudum) oil as a substitute for diesel engine oil in Thailand.

Journal of Agricultural Association of China, 120, 18.3. Mandpe, S., Kadlaskar, S., Degen, W., & Keppeler, S. (2005). On

road testing of advanced common rail diesel vehicles with bio-diesel from the Jatropha curcas plant (vol. 26, pp. 356364).Society of Automotive Engineering Inc.

4. Makkar, H. P. S., & Becker, K. (1997). Potential of Jatropha seedcake as protein supplement in livestock feed and constraints to its

utilization. In Proceedings of Jatropha 97: International sym-posium on biofuel and industrial products from Jatropha curcasand other tropical oilseed plants, Managua/Nicaragua, Mexico(pp. 2327).

5. Makkar, H. P. S., Aderibigbe, A. O., & Becker, K. (1998).

Comparative evaluation of non-toxic and toxic varieties of

Jatropha curcas for chemical composition, digestibility, proteindegradability and toxic factors. Food Chemistry, 62, 207215.

6. Martinez-Herrera, J., Sibdhiraju, S., Francis, G., Davila-Ortiz, G.,

& Becker, K. (2006). Chemical composition, toxic/antimetabolic

constituents and effects of different treatments on their levels, in

four provenances of Jatropha curcas L. from Mexico. FoodChemistry, 96, 8089.

7. Ginwal, H. S., Rawat, P. S., & Srivastava, R. L. (2004). Seed

source variation in growth performance and oil yield of Jatrophacurcas Linn in central India. Silve Senetica, 53, 186192.

8. Sujatha, M., Makkar, H. P. S., & Becker, K. (2005). Shoot bud

proliferation from axillary nodes and leaf sections of non-toxic

Jatropha curcas L. Plant Growth Regulations, 47, 8390.9. Francis, G., Edingger, R., & Becker, K. (2005). A concept for

simultaneous wasteland reclamation fuel production and socio

economic development in degraded areas in India need potential

& perspectives of Jatropha plantation. Natural Resources Forum,29, 1224.

10. Becker, K., & Makkar, H. P. S. (1998). Effects of phorbol esters

in carp (Cyprinus carpio L.). Veterinary Human Toxicology, 40,8289.

11. Sudheer, P. D. V. N., Singh, S., Mastan, S. G., Patel, J., & Reddy,

M. P. (2009). Molecular characterization and identification of

markers for toxic and non-toxic varieties of Jatropha curcas L.using RAPD, AFLP and SSR markers. Molecular BiologyReports, 36(6), 13571364.

12. Sudheer, P. D. V. N., Sarkar, R., Meenakshi, Boricha, G., &

Reddy, M. P. (2009). A simple protocol for isolation of high

quality genomic DNA from Jatropha curcas for genetic diversityand molecular marker studies. Indian Journal of Biotechnology,8, 187192.

13. Williams, J. G., Kubelik, A. R., Livak, J., Rafalski, J., & Tingey,

S. V. (1990). DNA polymorphism amplified by arbitrary primers

are useful as genetic markers. Nucleic Acid Resources, 18,65316535.

14. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecularcloning: A laboratory manual. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory Press.

15. Sudheer, P. D. V. N., Nirali, P., Reddy, M. P., & Radhakrishnan,

T. (2009). Comparative study of interspecific genetic divergence

and phylogenic analysis of genus Jatropha by RAPD and AFLP.

Molecular Biology Reports, 36, 901907.16. Jones, N., & Miller, J. H. 1991. Jatropha curcas a multipurpose

species for problematic sites. Land and Resources Series No. 1

(pp. 112). Washington: Asia Technical Department, World

Bank.

17. Heller, J. (1996). Physic nut-Jatropha curcas L. Promoting theconservation and use of underutilized and neglected crops.Rome: International Plant Genetic Resources Institute.

18. Gubitz, G. M., Mittelbach, M., & Trabi, M. (1999). Exploitation

of the tropical oil seed plant Jatropha curcas L. BioresourcesTechnology, 67, 7382.

19. Makkar, H. P. S., Becker, K., Sporer, F., & Wink, M. (1997).

Studies on nutritive potential and toxic constituents of different

provenance of Jatropha curcas. Journal of Agricultural FoodChemistry, 45, 31523157.

20. Makkar, H. P. S., Becker, K., & Schmoo, B. (1998). Edible

provenances of Jatropha curcas from Quinatana Roo state ofMexico and effect of roasting antinutrient and toxic factors in

seeds. Plant Foods for Human Nutrition, 52, 3136.21. Sudheer, P. D. V. N., Balaji, C., & Reddy, M. P. (2009). Genetic

diversity and phylogenetic analysis of genus Jatropha based on

nrDNA ITS sequence. Molecular Biology Reports, 36,19291935.

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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 DetectionAbstractIntroductionMaterials and MethodsGenomic DNA ExtractionRAPD AnalysisSCAR Marker IsolationMultiplex PCR Reaction for Identification of Non-Toxic J. curcas

Results and DiscussionSCAR Isolation and AmplificationMultiplex PCR

AcknowledgmentsReferences