jatropha intra population

Molecular characterization of intra-population variabilityof Jatropha curcas L. using DNA based molecular markers

Shaik G. Mastan Pamidimarri D. V. N. Sudheer

H. Rahman A. Ghosh Mangal S. Rathore

Ch. Ravi Prakash J. Chikara

Received: 21 January 2011 / Accepted: 3 May 2011 / Published online: 14 September 2011

Springer Science+Business Media B.V. 2011

Abstract Jatropha curcas L. (Euphorbiaceae) has

acquired a great importance as a renewable source of

energy with a number of environmental benefits. Very few

attempts were made to understand the extent of genetic

diversity of J. curcas germplasm. In the present study,

efforts were made to analyze the genetic diversity among

the elite germplasms of J. curcas, selected on the basis of

their performance in field using random amplified poly-

morphic DNA (RAPD), amplified fragment length poly-

morphism (AFLP) and simple sequence repeats (SSR). The

plants were selected on the basis of height, canopy cir-

cumference, number of seeds per fruit, weight of 100 seeds,

seed yield in grams per plant and oil content. Out of 250

RAPD (with 26 primers), 822 AFLP (with 17 primers) and

19 SSR band classes, 141, 346 and 7 were found to be

polymorphic, respectively. The percentage polymorphism

among the selected germplasms using RAPD, AFLP and

SSR was found to be 56.43, 57.9, and 36.84, respectively.

The Jaccards similarity coefficient was found 0.91, 0.90

and 0.91 through RAPD, AFLP and SSR marker systems,

respectively. Principle component analysis (PCA) and

dendrogarm analysis of genetic relationship among the

germplasm using RAPD, AFLP and SSR data showed a

good correlation for individual markers. The germplasm

JCC-11, 12, 13, 14 and 15 whose yield found to be high

were clustered together in dendrogram and PCA analysis

though JCC11 is geographically distinct from others. In

overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and

JCC 6 and JCC10 (in SSR) were found genetically diverse.

Characterization of geographically distinct and genetically

diverse germplasms with varied yield characters is an

important step in marker assisted selection (MAS) and it

can be useful for breeding programs and QTL mapping.

Keywords Jatropha curcas Diversity analysis Bio-diesel Marker assisted selection

Introduction

The current era of energy crisis, due to continuous deple-

tion of conventional sources of fuel and global warming,

rekindled the interest in promotion of non-conventional

sources of energy as an alternative. Plant borne oil is being

gaining importance as a viable option for the conventional

petro-diesel. A number of plant species have been

suggested as potential source and Jatropha curcas L.

(a member of Euphorbiaceae) is one among them. It is

native to South America and widely distributed in South

and Central America, Africa and Asia [1]. It is emerging as

an important source of bio-fuel because of its seed oil

which can be converted to biodiesel whose performance

demonstrated to be superior to petro-diesel. The short

gestation period, easy adaptation to different kinds of

marginal lands, drought endurance and avoidance by ani-

mals make the plant species more attractive. However, the

crop is characterized by variable and unpredictable yield

for reasons that have not been identified [2] which limit the

large scale cultivation and warrants need for genetic

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-011-1226-z) contains supplementarymaterial, which is available to authorized users.

S. G. Mastan P. D. V. N. Sudheer H. Rahman A. Ghosh M. S. Rathore Ch. Ravi Prakash J. Chikara (&)Discipline of Wasteland Research, Central Salt and Marine

Chemicals Research Institute, Council of Scientific and

Industrial Research, G.B. Badheka Marg, Bhavnagar,

Gujarat 364002, India

e-mail: [email protected]

123

Mol Biol Rep (2012) 39:43834390

DOI 10.1007/s11033-011-1226-z

improvement of the species. For the genetic improvement

of any species preliminary information about its genetic

back ground and characterized germplasm is very essential.

Molecular diversity analysis, germplasm characterization

through DNA fingerprinting techniques like random

amplified polymorphic DNA (RAPD), amplified fragment

length polymorphism (AFLP) and simple sequence repeats

(SSRs) have been well established and studied to some

extent in Jatropha to understand the extent of diversity that

exist; however, studies were limited only to natural popu-

lation and/or germplasms of narrow geographical area [3, 4].

Assessment of genetic diversity among elite germplasms

has important implications for breeding programs and

conservation of plant genetic resources. The characterized

germplasms and identified polymorphic markers are good

source of plant genetic resources and can be further

exploited for genetic improvement of the species through

marker assisted breeding and QTL analysis. Till date there

is very limited information about molecular characteriza-

tion of J. curcas germplasms, selected on the basis of

performance in field which is an important parameter for

genetic improvement of the plant species for yield attrib-

uting characters through marker assisted breeding. There-

fore, in present study attempts were made to assess genetic

diversity using molecular markers viz. RAPD, AFLP and

SSRs among different selected germplasms collected from

different geographical areas and whose performance is

evaluated in the field experiments.

Materials and methods

Plant materials and genomic DNA extraction

The plant material for present study was collected from 15

selected germplams of J. curcas growing in CSMCRI

experimental field station (Chorvadla, Gujarat at an altitude

21400N, 071470E). The plants were selected on the basisof their performance in the field trials (Table 1 supple-

mentary) viz. plant height, canopy circumference, seeds per

fruit, 100 seed weight, seed yield in grams per plant and oil

yield in percentage of the year 20082009. Genomic DNA

was extracted using CTAB protocol with slight modifica-

tion [5]. 0.1 g of leaf tissue was grinded in liquid nitrogen

and taken into a 2.0 mL microcentrifuge tube. To the

grinded tissues, 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 incu-

bated at 65C for 90 min. The samples were extracted withequal volume of chloroform:isoamyl alcohol (24:1) and

supernatant was transferred into a new tube. These samples

were treated with RNase and extracted with Tris saturated

phenol. The supernatant after extraction with Tris saturated

phenol was taken and extracted further with chloro-

form:isoamyl alcohol (24:1) twice, and precipitated with

80% of ethanol. The genomic DNA was air dried and

dissolved in 100 ll of Milli Q water. The genomic DNAwas quantified spectrophotometrically (Analytical spec-

trophotometer, U.K.) and diluted to the final concentration

of 1015 ng/ll.

RAPD analysis

Amplification of RAPD fragments was performed accord-

ing to Williams et al. [6], using decamer arbitrary primers

(Table 2 supplementary) (Operon technologies Inc, USA;

IDT, USA). The reaction was carried out in 25 ll volume ofreaction mixture containing final concentration of 10 mM

TrisHCl (pH 9.0), 50 mM KCl, 0.1 Triton X-100, 0.2 mM

each dNTPs, 3.0 mM MgCl2, 0.4 lM primer, 25 ng tem-plate, 1 U Taq DNA polymerase (Biogene, USA). Ampli-

fication was performed in programmed thermal cycler

(Master cycle epgradient S, eppendorf, Germany) with

program of initial denaturation at 94C for 3 min, 42 cyclesof denaturation at 94C for 30 s, primer annealing at 32Cfor 1 min, extension at 72C for 2.5 min and final extensionat 72C for 4 min. Amplification products were electro-phoresed in 1.5% agarose in 19 TBE buffer. The gels were

stained with ethidium bromide and documented using gel

documentation system (Syngene, UK).

AFLP analysis

AFLP fingerprinting was performed using AFLP analysis

system-II kit (Invitrogen Life Science Ltd., USA) accord-

ing to Vos et al. [7]. The genomic DNA (300 ng) was

digested with EcoRI and MseI at 37C for 2 h and digestedaliquot was ligated to EcoRI and MseI specific adopters at

20C for 90 min. The ligated DNA was diluted for 1:10and preamplified using EcoRI and MseI with one selective

nucleotide at the 30 end primer each. The preamplifiedproduct was diluted 1:10 with sterile TrisEDTA (TE)

buffer. The diluted products were amplified using different

combinations of EcoRI and MseI primer each with three

selective nucleotides at the 50 and 30, respectively (Table 3supplementary). Selective amplifications were performed

using 65C as the initial annealing temperature for the firstcycle and for subsequent 11 cycles the annealing temper-

ature was successively reduced by 0.7C. Twenty-threecycles were run at 56C annealing temperature. To thePCR product equal amount of formamide dye was added

and subjected to electrophoretic separation on 6% dena-

turing polyacrylamide gel in 19 TBE buffer in a

sequencing gel system (LKB, Sweden). The Gels were

stained with silver nitrate using silver staining kit (Sigma,

USA) and photographed for further recording.

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SSR analysis

Microsatellite amplifications were carried out in a volume

of 20 ll containing 0.25 U Taq DNA polymerase (Sigma,USA), 19 PCR buffer (10 mM TrisHCl, 50 mM KCl, 0.1

Triton X-100, pH 9.0), 0.2 mM dNTPs, 2 lM of eachprimer set, 2.8 mM MgCl2 and 50 ng template DNA.

Amplification cycle is consisted of an initial denaturation at

94C for 3 min, 35 cycles of denaturation at 94C for 15 s,specific annealing temperature for individual primer

(Table 4 supplementary) for 20 s, extension at 72C for30 s and final extension at 72C for 4 min. Amplificationproducts were electrophoresed in 8% polyacrylamide gel.

The gels were stained with ethidium bromide and docu-

mented using gel documentation system (Syngene, UK).

Data analysis

Experiment was repeated at least three times with each

primer and those primers gave reproducible fingerprints

were considered for data analysis. Acquired RAPD and

AFLP fingerprints were individually scored and statistically

analyzed by assuming the fragment size as biallelic (pres-

ent = 1, absent = 0) locus. A binary matrix was created.

Only those loci amplified strongly in each instance with

reproducibility were scored and included in the analyses.

Genetic similarity (GS) was calculated using Jaccards

coefficient of similarity [8] with the help of NTSYS-pc

package (version 2.2) [9]. In case of acquired SSR ampli-

fication, fingerprints were individually scored and statisti-

cally analyzed and obtained the genetic similarity based on

Nei and Li [10] definition as follows Sij = 2a/

(2a ? b ? c), where Sij is the similarity between two

individuals, i and j; a is number of bands present both in

i and j; b is number of bands present in i and absent in j; and

c is the number of bands absent in i and present in j. In all

cases (RAPD, AFLP and SSR analysis), the percentage of

polymorphism (PP) was calculated by using formula

PP = total number of polymorphic bands/total number of

bands multiplied with 100. Dendrograms were constructed

according to UPGMA (unweighted pair-group method with

arithmetic mean) method using binary data generated by

RAPD and AFLP followed by bootstrapping analysis across

the loci [11] with the help of NTSYS-pc software package.

Results

RAPD analysis

Out of 180 RAPD primers screened initially, 42 primers

produced amplification with more than 4 markers and were

included in present study for screening of germplasm. Out

of these 42 primers, 26 primers which produced clear bands

and reproducible fingerprints at each instance of repetition

were taken for the analysis (Fig. 1, Table 2 supplemen-

tary). With 26 primers, a total of 250 markers with an

average of 9.6 markers per primer were generated, out of

which 141 were polymorphic and remaining was mono-

morphic. The overall percentage of polymorphism among

germplasm was found 56.4. OPL7 produced the highest

number of markers [17] with 12 polymorphic loci with

70.59% of polymorphism. OPQ11 and opQ 20 generated

least number of markers [4], however opQ11 was found to

be 100% polymorphic. The number of polymorphic

markers varied 1 (opO2) to 12 (opL7) while percentage

polymorphism varied from 20% (opO2) to 100% (opQ11)

in the germplasm. Out of 26 primers used in present study,

15 produced more than 50% polymorphism in the

germplasm.

The average pair wise percentage polymorphism was

found 51.32%, ranging from 6.05 (JCC13 and JCC14) to

31.53 (JCC6 and JCC10) (Table 5 supplementary data). In

present study JCC6 was found to be the most diverged

genotype among the studied germplasm. In pair wise

comparison, average Jaccard coefficient of genetic simi-

larity was found 0.90, ranging from 0.81 (JCC6 and

JCC10) to 0.97 (JCC13 and JCC14) (Table 6 supplemen-

tary data). Three major clusters were obtained in RAPD

dendrogram (Fig. 2). In RAPD dendrogram, least genetic

distance was observed between JCC13 and JCC14; and it

was followed by JCC1 and JCC2. JCC6 showed highest

genetic divergence from rest of the genotypes and it was

followed by JCC10. Both of them stand separate individ-

ually as well as from rest of the population in RAPD

dendrogram. PCA analysis of RAPD data resulted in

identification of four distinct groups, consisting of 1, 3, 3

and 8 genotypes. JCC6 stand separate from all the geno-

types, which is in accordance to RAPD dendrogram, while

JCC10 clustered with largest group in RAPD-PCA analysis

(Fig. 3).

Fig. 1 RAPD finger printing of selected J. curcas germplasm usingprimer opQ9; 115 JCC1JCC15 and M 1 kb marker

Mol Biol Rep (2012) 39:43834390 4385

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AFLP analysis

Out of 21 selective primer combinations, 17 primers

producing reproducible fingerprint (Table 3 supplemen-

tary) with more than 30 markers in each instance of

repetition were used for data analysis (Fig. 4). A total of

822 AFLP markers with an average of 48.35 per primer

combination were obtained, out of these 346 were poly-

morphic. Numbers of amplicon ranged from 33 (P64) to

68 (P12). The overall polymorphism among germplasm

was found to be 57.9%. Highest (69.57%) polymorphism

was recorded with P11 primer combination with highest

(32) polymorphic markers while P35 reported least

(5.26%) polymorphism with least [2] number of poly-

morphic markers.

In pair wise comparison among germplasm, average

polymorphism was found to be 15.32% ranging from 3.05%

(JCC12 and JCC13) to 37.60% (JCC1 and JCC8) (Table 7

supplementary data). JCC8 was found to be the most

diverged genotype while JCC14 was least diverged accord-

ing to pair wise comparisons of AFLP data. The Jaccard

coefficient of genetic similarity ranged from 0.77 (between

JCC8 and JCC1) to 0.98 (between JCC13 and JCC12) with

an average of 0.91(Table 8 supplementary data). In den-

drogram constructed on the basis of AFLP data, a single

major cluster was obtained, which included all the genotypes

except JCC1, JCC6 and JCC8. JCC8 showed highest genetic

divergence in AFLP analysis and it was followed by JCC6

and JCC1, respectively (Fig. 5). Least genetic divergence

was found between JCC12 and JCC13; and it was followed

Fig. 2 Dendrogram showinggenetic relationship with

bootstrapping values among the

selected germplasm of J. curcasthrough RAPD

Fig. 3 PCA (principlecomponent analysis) showing

the genetic relationship among

the selected germplasm of

J. curcas through RAPD

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by JCC2 and JCC3; JCC12/13 and JCC14. In AFLP-PCA

analysis, one major group was obtained consisting of most of

the genotypes except JCC6 and JCC8, standing individually

as accordance to the AFLP dendrogram (Fig. 6).

SSR analysis

SSR amplification was performed with primers designed

for twenty-five markers (twenty SSR from Sudheer et al.

[12] and five from Sun et al. [13]. Out of these, 19 primers

with reproducible results at every instance of repetition and

used for data analysis (Fig. 7). The size of loci with these

primers ranged from 92 to 459 bp (Table 4 supplemen-

tary). Seven loci out of 19 were found polymorphic. The

number of alleles at these polymorphic loci ranged from 2

(JCMNS 183 and JCDS 24 primers) to 5 (JCPS 7 and

JCMNS 292 primer). Overall 36.8% loci were found

polymorphic among the studied germplam. Average Jac-

card coefficient was found 0.91, ranging from 0.78

(between JCC8 and JCC10) to 1.00 (between JCC1 and

JCC11; JCC12 and JCC14) (Table 9 supplementary data).

Three major clusters were obtained in SSR dendrogram

(Fig. 8). JCC1, 11, 5, 12, 14, 15, 13 and 7 clustered toge-

ther however belong to different geographical area. JCC2

and JCC3 found to be diverse however they belong to the

same geographical area. JCC1 and JCC11; JCC12 and

JCC14 showed least genetic distance while JCC6 and

JCC10 were found highly diverse from rest of the popu-

lation; however they formed same cluster. SSR PCA

analysis resulted in formation of two major groups, while

JCC2, 6, 3, 8 and 10 remained individually (Fig. 9).

Discussion

Molecular characterization of cultivars for the investigation

of genetic diversity and to confirm the uniformity, stability

and distinctness of different cultivars accelerated their

application in molecular breeding for the improvement of

the species [14]. Unlike the morphological and enzymatic

markers whose variations can occur due to the environ-

mental fluctuations, the molecular marker will be stable

and reproducible [15, 16]. Thus the characterized

Fig. 4 AFLP finger printing profle of Elite J. curcas germplasmusing primer E-ACT/M-CAG; 115 JCC1JCC15 and M 1 kb marker



selected germplasm of J. curcasthrough AFLP

Mol Biol Rep (2012) 39:43834390 4387

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germplasm and the identified markers can be a good source

of plant genetic resources and can further be exploited for

genetic improvement of the species through marker assis-

ted breeding to obtain the better variety with high yielding

and better performing attributes. In present study all the

marker systems being employed to analyze the intra-pop-

ulation diversity of J. curcas germplasm were quite

informative and were able to generate unique DNA fin-

gerprints and adequate polymorphism among the germ-

plasm under investigation.

Beside tremendous economic benefits, there were very

few studies carried to understand the genetic diversity

using various marker systems in J. curcas. Basha and Su-

jatha 2007 [3] studied the extent of genetic diversity among

toxic and non-toxic varieties using RAPD and the per-

centage of GS is found to be 96.3. In another study Sudheer

et al. 2008 [17] reported 84.91 and 83.59% (GS) among

toxic and non-toxic J. curcas by RAPD and AFLP,

respectively and identified the specific markers of RAPD

and AFLP for both the varieties. Inter and intra-population

studies using RAPD and ISSR in 42 germplasm of

J. curcas collected from different regions in India along

with a non-toxic genotype from Mexico showed 42.00 and

37.40 PP by RAPD and ISSR, respectively [3]. Sudheer

et al. [17] studied 9.72 and 20.57 percent polymorphism in

natural germplasm of J. curcas using RAPD and AFLP,

respectively. Though there are a number of studies on

diversity analysis in J. curcas, however till date no sys-

tematic studies were made on the analysis of genetic

diversity among the selected germplasm; whose perfor-

mance was evaluated in the field. Therefore, the present

study was conducted to evaluate the genetic diversity

among the selected germplasm whose yield attributing

characters were evaluated.

In India extensive experimental field trials are being

conducted at CSMCRI (Central Salt and Marine Chemical

Research Institute) to assess the various factors like vari-

ability in morphology, seed yield, and oil content, tolerance

to biotic and abiotic stress in natural population. In the

present study taking the consideration of the yield




J. curcas through AFLP

Fig. 7 SSR profle of Elite J. curcas germplasm using primerJCMNS-292; 115 JCC1JCC15 and M 1 kb marker

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attributing characters selected germplasm were taken for

the assessment of the molecular diversity. The mean

genetic similarity observed was found to be 0.91, 0.90 and

0.91 by RAPD AFLP and SSR, respectively which is in

accordance with previous reports [4, 18]. The dendrogram

analyses of germplasm using the three molecular markers

have given a good correlation of genetic similarity among

them. The PCA analysis of these markers showed good

correlation with dendrogram of respective marker system.

In dendrogram and PCA analysis of RAPD, AFLP and SSR

markers, JCC6 is found to be highly diverse from rest of

population however it was found to be clustered with

JCC10 in SSR dendrogram analysis.

Though JCC6, 12, 13, 14 and 15 were collected from

same geographical locations (i.e. Gujarat) and possess quite

good yield attributing characters, these germplasm clustered

together in the dendrograms and PCA analysis of all the

three marker systems except JCC6. JCC11 collected from

Orissa having highest yield (33.24% oil content) clustered

with JCC12, 13, 14, 15 (Gujarat) in RAPD, AFLP and SSR

dendrogram and also correlated with the PCA analysis of

RAPD, AFLP and SSR irrespective of geographical area.

JCC1, 2, 3, 4, 5, 6, 10, 12, 13, 14 and 15 belong to the same

geographical area (Gujarat), however they differ in yield

attributing and molecular characters. In the dendrogram

analysis JCC1, 2 and 3 clustered in the same but JCC4 found

to be in the separate cluster showing the genetic distinctness

from JCC1, 2 and 3 irrespective of geographical area. These

differences in molecular characters irrespective of geo-

graphical areas might be due to the anthropogenic activity as

reported in the previous studies [3, 4].

In the present study though the overall genetic diversity

found to be less in comparison with previous studies,

however the mean percentage of polymorphism is found to

be in accordance with Sudheer et al. [4, 17] and Basha and

Sujatha [3]. The type of genetic polymorphism, use of

different marker systems and the number of primers used

affect the correlations among different markers [19].



selected germplasm of J. curcasthrough SSR




J. curcas through SSR

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Similarly the degree of genetic polymorphism detected in

the selected germplasm may be affected due to the three

marker systems and the number of primers used for study.

Variation of diversity among the germplasm using three

markers in the present study may be due to the codominant

nature of SSRs and dominant nature of RAPD and AFLP

markers. As the AFLP gives more amplified fragments than

RAPD followed by SSRs, it shows highest polymorphism

when compared with others. The finding of a slightly

higher resolution of genetic similarities by RAPDs and

AFLPs, compared to SSRs, may be due to the high poly-

morphism of SSRs which render them less suitable for

determining genetic relationships among cultivars [20].

The present study of diversity analysis revealed by

molecular markers and yield attributing characters among

selected germplasm can be useful in further breeding pro-

grams for generation of hybrids, in maintenance of selected

genetic stocks and for molecular ecological studies and

also further will pave way for the creation of mapping

population and linkage analysis, marker assisted selection

and QTL analysis for improvement of the species for its

yield attributing characters.

Acknowledgments The authors wish to thank Council for Scientificand Industrial Research (CSIR), New Delhi, India for financial

support.

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Molecular characterization of intra-population variability of Jatropha curcas L. using DNA based molecular markersAbstractIntroductionMaterials and methodsPlant materials and genomic DNA extractionRAPD analysisAFLP analysisSSR analysisData analysis

ResultsRAPD analysisAFLP analysisSSR analysis

DiscussionAcknowledgmentsReferences

jatropha intra population

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