Molecular characterization and genetic diversity analysisof Jatropha curcas L. in India using RAPD and AFLP analysis

D. V. N. Sudheer Pamidimarri Shaik G. Mastan Hifzur Rahman Muppala P. Reddy

Received: 16 March 2009 / Accepted: 3 August 2009 / Published online: 18 August 2009

Springer Science+Business Media B.V. 2009

Abstract Jatropha curcas L. belongs to family Euphor-

biaceae, native to South America and widely distributed in

South and Central America, attained significant importance

for its seed oil which can be converted to biodiesel, a

renewable energy source alternative to conventional petro-

diesel. Very few attempts were made to understand the

extent of genetic diversity that exists in J. curcas. There-

fore, the present investigation was undertaken to asses the

genetic diversity among 28 diverse germplasm collected

from distinct geographical areas in India. The overall

percentage of polymorphism (PP) was found to be 50.70

and 60.95 by RAPD and AFLP, respectively. The mean PP

was found to be 9.72 and 20.57 by RAPD and AFLP,

respectively. The mean genetic similarity was observed to

be 0.89 by RAPD and 0.88 by AFLP. Among the germ-

plasm JCI20 found to be the most diverged one. The

dendrogram analysis of RAPD and AFLP data showed

good congruence, but better resolution and more poly-

morphism was observed with AFLP. When the dendrogram

of RAPD was compared with AFLP dendrogram, the major

clustering pattern was found to be similar; however,

changes in minor grouping were observed. In both RAPD

and AFLP analysis clustering of germplasm did not show

any correlation with the geographical area of collection.

Low genetic diversity observed in J. curcas and the

clustering pattern indicates that the distribution of species

might have happened through anthropogenic activity and

warrants the need for widening the genetic base. The

present study will provide pavement for further intra-pop-

ulation studies on narrow geographical areas, to understand

the population genetic structure, phylogeography, molec-

ular ecological studies. The marker information and the

characterized germplasm help in further improvement of

the species through marker assisted breeding programs.

Keywords AFLP (amplified fragment length

polymorphism) Biodiesel Genetic diversity Jatropha curcas Molecular markers RAPD (random amplified polymorphic DNA)

Introduction

In the current era of energy crisis due to the depletion of

natural fuel resources and global warming, Jatropha curcas

has acquired notable importance as an alternative renew-

able energy source [1, 2] and offer prospects by increasing

energy supplies in a self reliant way in developing coun-

tries like India and also work as checkpoint for aggravating

green house gases [3]. Biodiesel derived from J. curcas

seed oil has the desirable physiochemical characteristics,

performance was demonstrated to be superior to conven-

tional petro-diesel [1, 2, 4, 5] and has created a surge of

interest in cultivation of this species all over the world.

J. curcas the most primitive species of genus Jatropha [6]

is native to South America and widely distributed in South

and Central America, Africa, and Asia and has the ability

to rehabilitate the waste and degraded lands [7]. Easy

adaptation to different kinds of marginal lands, drought

endurance, avoidance by animals and its short interval time

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-009-9712-2) contains supplementarymaterial, which is available to authorized users.

D. V. N. S. Pamidimarri S. G. Mastan H. Rahman M. P. Reddy (&)Discipline of Wasteland Research, Central Salt and Marine

Chemicals Research Institute (CSIR), Bhavnagar, Gujarat

364002, India

e-mail: mpr@csmcri.org

123

Mol Biol Rep (2010) 37:22492257

DOI 10.1007/s11033-009-9712-2

to give first yield make this plant species more attractive

for cultivation. The species can grow even in areas with

extreme climate and soil conditions that could not be

inhabited by most of the agriculturally important plant

species which circumvent the food versus fuel resentment

[3]. However, the crop is characterized by variable and

unpredictable yield for the reasons that have not been

identified [8] which is limiting the large scale cultivation

and warrants need for genetic improvement of the species.

For the genetic improvement of any species information

about its genetic back ground and characterized germplasm

is very essential.

Molecular diversity analysis, germplasm characteriza-

tion through DNA fingerprinting techniques like RAPD

and AFLP have been well established and studied to some

extent in Jatropha to understand the extent of diversity that

exist. Recent phylogenetics studies in genus Jatropha

reveled using RAPD, AFLP and nrDNA ITS sequence [9]

found that J. curcas is genetically more similar with spe-

cies Jatropha integerrima than any other species of genus

Jatropha. The studies were carried to characterize the toxic

and non-toxic varieties of J. curcas using RAPD and

AFLP, respectively and inter and intra-population studies

using RAPD, ISSR and AFLP in India [10, 11]. However,

these studies were limited to two different varieties, pop-

ulation and/or germplasm of narrow geographical areas

only [7, 10, 11].

Therefore, the present investigation was undertaken to

characterize the germplasm collected from distinct geo-

graphical areas of India to asses the genetic diversity and to

deduce the genetic relationships among the germplasm

with the help of clustering analysis. This study will provide

the genetic back ground of J. curcas and the extent of

molecular diversity exist among the germplasm. The

characterized germplasm and identified polymorphic

markers can be a good source of plant genetic resources

and can be further exploited for genetic improvement of the

species through marker assisted breeding.

Materials and methods

Genomic DNA extraction

Genomic DNA was extracted using CTAB protocol as

described by Sudheer et al. [12] from 28 diverged germ-

plasm (Table 1, Fig. 1) established in Central Salt and

Marine Chemicals Research Institute, Bhavnagar, Gujarat,

India, experimental field (21750N, 72140E). About 0.1 gof leaf tissue was ground in liquid nitrogen and taken in to

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. Theabove sample was extracted with equal volume of chloro-

form: isoamyl alcohol (24:1) and supernatant was trans-

ferred in to a new tube. The sample was treated with RNase

and extracted with Tris saturated phenol. The supernatant

after extraction with Tris saturated phenol was taken and

extracted further with chloroform: isoamyl alcohol (24:1)

twice, and precipitated with 80% of ethanol. The pellet was

air dried and dissolved in 100 ll of Milli Q water.In our earlier studies both RAPD and AFLP systems

were found equally competent [7, 13] and the same marker

systems have been used in the present study.

RAPD analysis

Amplification of RAPD fragments was performed accord-

ing to Williams et al. [14] 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 (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 template, 1 unit TaqDNA polymerase (Sigma, USA). Amplification was per-

formed in programmed thermal cycler (Master cycle ep-

gradient S, eppendorf, Germany) with program of initial

denaturation at 94C for 3 min, 42 cycles of denaturationat 94C for 30 s, primer annealing at 32C for 1 min,extension at 72C for 2.5 min and final extension at 72Cfor 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 done three times with each primer

and those primers gave reproducible fingerprints were

considered for data analysis.

AFLP analysis

AFLP analysis system-II kit (Invitrogen life science Ltd,

USA) was used for AFLP fingerprinting [15]. The genomic

DNA (300 ng) was digested with EcoRI and MseI at 37Cfor 2 h and digested aliquot was ligated to EcoRI and MseI

specific adaptors at 20C for 90 min. The ligated DNA waspreamplified using EcoRI and MseI with one selective

nucleotide at the 30 end primer each. The preamplifiedproduct was diluted 1:20 with sterile TE buffer. The diluted

product was amplified with selective primer EcoRI and

MseI having three selective nucleotides at the 30 end. PCRwas performed using 65C as the initial annealing tem-perature for the first cycle and for subsequent 11 cycles the

annealing temperature was successively reduced by 0.7C.Twenty-three cycles were run at 56C annealing tempera-ture. To the PCR product equal amount of formamide dye

2250 Mol Biol Rep (2010) 37:22492257

123

was added and subjected to electrophoretic separation on

6% denaturing 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). Experiment with each primer combination of EcoRI,

MseI was done three times and those combinations gave

reproducible fingerprints were considered for data analysis.

Data analysis

Generated RAPD and AFLP fingerprints were individually

scored and statistically analyzed by following the

assumption that fragment size as a locus was considered as

biallelic (present = 1, absent = 0) and made the binary

matrix. Only those loci amplified strongly in each instance

with reproducibility were scored and included in the

analyses ignoring the intensity of the band. Genetic simi-

larity (GS) was calculated using Jaccards coefficient of

similarity [16] with the help of NTSYS-pc package

(version 2.2) [17]. 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 [18] with

the help of NTSYS-pc software.

Result

RAPD analysis

Initially 180 RAPD primers (20 primers Kit E, IDT USA;

160 primers, kit-J, K, L, N, O, P, Q, R, Operon technolo-

gies Inc., USA) were screened and 52 primers responded

with more than six markers were included in the study. Out

of 52 RAPD primers screened 29 responded with band

Table 1 Details of thegeographic locations of J.curcas gerplasm used in thestudy

Serial

number

Germplasm

code

State provenance

of collection (India)

Latitude Longitude

1 JCI01 Orissa 218228N 8.5808.680E2 JCI02 Gujarat 22, 74.38N 75, 57.8463 JCI03 Gujarat 21560, 18.45 72100, 43.844 JCI04 Uttar Pradesh 26.45N 83.23E

5 JCI05 Uttar Pradesh 25.57N 81.50

6 JCI06 Gujarat 24, 16.35 72, 44.937 JCI07 Gujarat 21.50, 17.53 72.10, 44.968 JCI08 Gujarat 22, 17.350 75, 57.420

9 JCI09 Gujarat 24, 17.430 72, 46.930

10 JCI10 Orissa 9, 25.440 84, 56.820

11 JCI11 Orissa 19, 23.360 84, 56.420

12 JCI12 Orissa 19, 27.420 85, 2.080

13 JCI13 Arunachal Pradesh 27000 0 93420 0

14 JCI14 Madhya Pradesh 22.59, 38.1 75.89, 83.4

15 JCI15 Assam 26730 0 94010 0

16 JCI16 Andhra Pradesh 18.3N 83.3E17 JCI17 Bihar 25.30N 85.14E

18 JCI18 Tamil Nadu 11.190, 141 76.560, 16519 JCI19 Chhattisgarh 21.13N 81.41E

20 JCI20 Jharkhand 23.19N 85.27E

21 JCI21 Gujarat 24, 18.940 72, 46.320

22 JCI22 Rajasthan 24 42 03 73 38 13

23 JCI23 Karnataka 1735018250N

7642077390E

24 JCI24 Maharashtra 204202260N 77207760E25 JCI25 Tamil Nadu 11.040, 327 76.490, 70226 JCI26 Haryana 28.27N 77.01E

27 JCI27 Kerala 11.050, 538 76.370, 52928 JCI28 Andhra Pradesh 17420N 83240E

Mol Biol Rep (2010) 37:22492257 2251

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clarity and reproducibility were taken for final analysis

(Table 2). Overall PP was found to be 50.70. On average

the primers studied resulted with 10.59 loci containing 5.37

polymorphic and 5.22 non polymorphic loci. The mean PP

given by primers studied was observed to be 52%. The PP

given by each primer ranged from 10 (OPL5) to 85.71

(OPN8) (Table 2). Though the use of OPN 8 primer

resulted in maximum polymorphism (85.71), better reso-

lution was observed with IDT E5 and OPN 4 primers. IDT

E 4, 5, 18, OPN 4, 8, 19, OPO18, 19, OPP2, 6 OPQ7 and

15 primers produced more than 60% of polymorphism. Out

of 28 germplasm studied minimum PP was recorded

between JCI01 and JCI02 and maximum between JCI15

and JCI17. JCI17 showed highest PP when compared with

other germplasm studied. PP by pair wise comparison

between any two germplasm ranged from 0.20 to 24.01

(Table 4) with mean PP 9.72. When JCI01 was compared

with JCI02 genetic diversity was negligible. Out of 248

markers scored between JCI01 and JCI02 only one poly-

morphic marker (820 base pair) was found with IDT E 12

primer and minimum GS (0.76) was observed when JCI15

was compared with JCI17. Overall mean GS was found to

be 0.89. The maximum pair wise GS (1.00) was found

between JCI01 and JCI02, and the minimum (0.77)

between JCI05/JCI17; JCI06/JCI17 and JCI10/JCI17

(Tables 4 and 5, supplementary material).

The dendrogram was constructed according to UPGMA

using binary data of RAPD and resulted dendrogram

showed two major clusters (Fig. 2). The cluster I was

formed with 15 germplasm of six state provenances; Guj-

arat (JCI02, 06, 07, 08, 09, 03), Orissa (JCI01, 12, 11, 10),

Utter Pradesh (JCI04, 05), Arunachal Pradesh (JCI13),

Madhya Pradesh (JCI14), Assam (JCI15). Cluster II was

included with eleven state provenances; Andhra Pradash

(JCI16, 28), Tamilnadu (JCI18, 25), Gujarat (JCI21),

Chattisgarh (JCI19), Rajasthan (JCI22), Karnataka (JCI23),

Table 2 List of RAPD primers,number of amplified products

and percentage of

polymorphism obtained by

analyzing 28 Indian germplasm

of J. curcas L

Primer Sequence of

RAPD

primer 50-30

Number of

polymorphic

markers

Number of

nonpolymorphic

markers

Percentage of

polymorphism

IDT E 5 TCAGGGAGGT 10.00 4.00 71.43

IDT E 12 TTATCGCCCC 11.00 10.00 52.38

IDT E 4 GTGACATGCC 10.00 6.00 62.50

IDT E 7 AGATGCAGCC 6.00 11.00 35.29

IDT E 18 GGACTGCAGA 8.00 4.00 66.67

OPJ13 CCACACATACA 3.00 8.00 27.27

OPJ20 AAGCGGCCTC 6.00 10.00 37.50

OPL5 ACGCAGGCAC 1.00 9.00 10.00

OPN4 GACCGACCCA 7.00 3.00 70.00

OPN7 CAGCCCAGAG 1.00 3.00 25.00

OPN8 ACCTCAGCTC 6.00 1.00 85.71

OPN12 CACAGACACC 5.00 5.00 50.00

OPN14 TCGTGCGGGT 3.00 8.00 27.27

OPN16 AAGCGACCTG 6.00 6.00 50.00

OPN19 GTCCGTACTG 6.00 2.00 75.00

OPN20 GGTGCTCCGT 2.00 8.00 20.00

OPO2 ACGTAGCGTC 3.00 2.00 60.00

OPO5 CCCAGTCACT 5.00 4.00 55.56

OPO18 CTCGCTATCC 5.00 3.00 62.50

OPO19 GGTGCACGTT 6.00 3.00 66.67

OPO20 GGTGCACGTT 3.00 3.00 50.00

OPP2 TCGGCACGCA 4.00 2.00 66.67

OPP6 GTGGGCTGAC 4.00 2.00 66.67

OPP14 CCAGCCGAAC 2.00 2.00 50.00

OPP15 GGAAGCCAAC 4.00 3.00 57.14

OPQ7 CCCCGATGGT 7.00 3.00 70.00

OPQ9 GGCTAACCGA 4.00 10.00 28.57

OPQ15 GGGTAACGTG 4.00 2.00 66.67

OPQ2 TCTGTCGGTC 3.00 4.00 42.86

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Kerala (JCI27), Maharashtra (JCI24), Haryana (JCI26),

Bihar (JCI17) and Jharkhand (JCI20). Among the germ-

plasm analyzed JCI01 and JCI02 showed lowest genetic

distance in the dendrogram though their geographical area

of collection is far apart. Half of the germplasm collected

from Gujarat (JCI 07, 08, 09) and Orissa (JCI10, 12)

clustered together and formed a sub cluster in major cluster

I and they are intern clustered with germplasm of Utter

Pradesh (JCI04, 05) Orissa (JCI01) and Gujarat (JCI02).

The cluster II when compared to cluster I included with

more diverse germplasm of various state provenances.

AFLP analysis

Using 18 combinations of AFLP selective primers which

resulted in superior quality fingerprints with above 30

markers were taken for analysis. On average the primers

studied resulted in 51.22 markers with 41 markers poly-

morphic. Total 950 markers were included in the analysis

and 579 markers found to be polymorphic. On an average

the primers studied resulted with 52.78 loci containing

32.17 polymorphic and 20.61 nonpolymorphic loci. Over-

all average PP given by primers was 61.24. The extent of

Fig. 2 Dendrogram generatedusing UPGMA for 28

germplasm of J. curcas basedon RAPD data obtained by 29

primer combinations. The

values on the nodes of thecluster indicate the bootstrap

values and the scale representsJaccards similarity coefficient

values

Fig. 1 Map displaying thelocation of J. curcas germplasmcollected in India

Mol Biol Rep (2010) 37:22492257 2253

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polymorphism varied from 76.32% by primer set P33 to

47.14% by primer set P52. Among the 18 primers studied

highest number of polymorphic markers (42) was resulted

with the primer combination P5. Though the primer set P33

resulted with maximum PP, but produced lowest number of

markers among the primer sets used in the present study

(Table 3). Maximum GS (0.98) and minimum PP (4.01)

was seen between the germplasm JCI07 and JCI08. Mini-

mum GS (0.70) and maximum PP (45.79) was found

between JCI15 and JCI21. Overall PP was found to be

60.95, and the mean PP between any two germplasm was

found to be 20.57. The mean GS between the germplasm is

found to be 0.88. Maximum pair wise GS (0.98) was found

between the germplasm JCI07/JCI08. The minimum (0.71)

was found when JCI20 is compared with JCI05, 06, 07, 08,

09, and 11(Tables 6 and 7, supplementary material).

The dendrogram (Fig. 3) of AFLP data also showed two

major clusters and cluster I is included with the germplasm

of six state provenances; Orissa (JCI01, 10, 11, 12), Gujarat

(JCI02, 03, 07, 08, 09, 06), Uttar Pradesh (JCI04, 05),

Arunachal Pradesh (JCI13), Madhya Pradesh (JCI14) and

Assam (JCI15). Cluster II is included with germplasm

belongs to eleven state provenances; Andhra Pradesh

(JCI16, 28), Bihar (JCI17), Tamilnadu (JCI18, 25), Gujarat

(JCI21), Chhattisgarh (JCI19), Rajasthan (JCI22), Karna-

taka (JCI23), Maharashtra (JCI24), Haryana (JCI26), Ker-

ala (JCI27) and Jharkhand (JCI20). As in RAPD

dendrogram, germplasm JCI20 separated from rest of the

samples in the major cluster II and formed separate branch

even in AFLP. As in RAPD dendrogram, major cluster II of

AFLP dendrogram also included with the germplasm

belong to more distinct geographical areas. Similar to

RAPD results the germplasm collected from Gujarat

(JCI02, 03, 06, 07, 08, 09) and Orissa (JCI01, 10) grouped

together in cluster I. Similarly, JCI10, 09, 08, 07 those

clustered together in RAPD dendrogram also clustered

together in AFLP dendrogram. In the same way germplasm

JCI 03, 04 and 05 also grouped together in both RAPD and

AFLP dendrogram (Figs. 2, 3).

Discussion

Many studies were carried out to analyze the diversity within

and among the population of plant species using allozymes

[19, 20]. The limitations with these techniques were low

number of markers and pseudo variations [2025]. Advan-

ces in the field of molecular biology have provided many

tools for studying the diversity in genome level to get genetic

relationship within and among the species. Out of many PCR

based fingerprinting techniques RAPD and AFLP emerged

as very useful and efficient methods for analyzing the

molecular diversity due to ease of work and efficiency in

utilizing for the study of divergence among different plant

species [12, 26, 27], within the species [28, 29], varieties [9].

Jatropha curcas is a small genome species having the

2C genome of 0.85 pg size with 38.7% of average GC base

composition. The karyotype of J. curcas is made up of 22

Table 3 List of AFLP selectiveprimer combination, number of

amplified products and

percentage of polymorphism

obtained by analyzing 28 Indian

germplasm of J. curcas L

Selective primer

combination

Number of

polymorphic

markers

Number of

nonpolymorphic

markers

Percentage of

polymorphism

P1(MCAA/EAAC) 39.00 15.00 72.22

P4(MCAT/EAAC) 46.00 22.00 67.65

P5(MCTA/EAAC) 42.00 33.00 56.00

P6(MCTC/EAAC) 37.00 19.00 66.07

P7(MCTG/EAAC) 28.00 21.00 57.14

P11(MCAG/EAAG) 32.00 22.00 59.26

P12(MCAT/EAAG) 27.00 22.00 55.10

P18(MCAC/EACA) 14.00 20.00 41.18

P19(MCAG/EACA) 32.00 10.00 76.19

P20(MCAT/EACA) 32.00 15.00 68.09

P27(MCAG/EACT) 32.00 20.00 61.54

P29(MCTA/EACT) 29.00 18.00 61.70

P33(MCAA/EACC) 29.00 9.00 76.32

P34(MCAC/EACC) 40.00 26.00 60.61

P39(MCTG/EACC) 28.00 28.00 50.00

P50(MCAC/EAGC) 34.00 16.00 68.00

P52(MCAT/EAGC) 33.00 37.00 47.14

P54(MCTC/EAGC) 25.00 18.00 58.14

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relatively small metacentric and submetacentric chromo-

somes whose size range from 1.71 to 1.24 lm [30]. Phy-logenetic analysis using RAPD, AFLP and nrDNA ITS

sequence concluded that J. curcas is genetically more close

to J. integerrima than any other species of the genus

Jatropha and reported to be the reason for successful intra-

specific hybridization [12].

There were very few studies carried to understand the

diversity using various marker systems in J. curcas. Suja-

tha et al. [31] 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. [7] 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 [10]. Ram et al. [32] char-

acterized five accessions from one region with 18 RAPD

primers and Ranade et al. [33] subjected 22 accessions

from six regions using 7 RAPD and four directed ampli-

fication of minisatellite DNA (DAMD) primers. Novel

microsatellites isolated from J. curcas and were charac-

terized in a population showed that showed significant

deviation from HardyWeinberg equilibrium may be due

to anthropogenic activity in distribution of the population

[34] and correlates with the present results. The RAPD and

AFLP markers were also characterized in toxic and non-

toxic varieties of J. curcas and 7 out of 12 markers showed

size polymorphism [7]. The comparative analysis of inter

and intra-specific diversity analysis of genus Jatropha by

RAPD and AFLP indicated existence of low genetic

diversity in J. curcas [12] and the similar observation was

reported [10, 11]. Tatikonda et al. [11] studied the diversity

among the germplasm collected from six states and the

results were in agreement with our previous studies [12].

Genetic diversity analyses studies in J. curcas were

limited to some varieties and group of populations and/or

germplasm of narrow geographical area only [7, 10, 11]. By

establishing genetic distances of germplasm of distinct

geographical areas and genetic diversity analysis gives

molecular ecology of that species in that area and charac-

terized germplasm can be very efficiently used for genetic

improvement of the species through marker based breeding

programs [10, 12]. In our previous study with multilocus

marker systems RAPD and AFLP, we found both the

marker systems were equally competent [7, 10] and the

same marker systems have been selected for molecular

characterization, estimation of genetic diversity for 28

germplasm belonging to distinct geographical areas of

India. Hence, the present study was taken to understand the

extant of genetic diversity of J. curcas and to deduce their

genetic relationships using RAPD and AFLP fingerprinting.

The present comparative study of RAPD and AFLP

analysis with 28 diverged germplasm collected from dis-

tinct geographical area showed good correlation in genetic

relatedness and clustering; however, AFLP showed supe-

rior marker polymorphism better resolution in the den-

drogram analysis. PP in the present study showed 50.70

and 60.95% by RAPD and AFLP, respectively. The supe-

rior PP observed in this study as compared to earlier studies

may be due to inclusion of germplasm of more distinct

geographical areas. Polymorphism obtained with AFLP

fingerprinting (60.95%) was higher than other marker

systems. In comparison with many species, the genetic

diversity in J. curcas is far less [35, 36] and this narrow

genetic diversity observed may be because of few intro-

ductions of the species and further distribution of the race

through vegetative propagation and by anthropogenic

activity as viewed in the earlier studies [7, 10].

Fig. 3 Dendrogram generatedusing UPGMA for 28

germplasm of J. curcas basedon AFLP data obtained by 18

primer combinations. The

values on the nodes of thecluster indicate the bootstrap

values and the scale representsJaccards similarity coefficient

values

Mol Biol Rep (2010) 37:22492257 2255

123

Although the germplasm were not clustered in accor-

dance to the geographical areas, the clustering pattern of

the genotypes in both the dendrograms of RAPD and AFLP

showed good congruence. Higher bootstrap values (above

50) obtained for nodes in dendrograms indicate the con-

sistency of grouping the germplasm in different clusters. In

case of RAPD dendrogram among the germplasm analyzed

JCI01 and JCI02 showed lowest genetic distance though

their geographical area of collection was far apart. Simi-

larly germplasm collected from Gujarat (JCI 07, 08, 09)

and Orissa (JCI10, 12) clustered together and sub clustered

in the cluster I. Among the germplasm studied no corre-

lation in their clustering pattern was observed with geo-

graphical area of collection. Similarly JCI03, 11, 21

collected from Gujarat, Orissa, respectively; separated

from the sub cluster formed by the germplasm collected

from Gujarat and Orissa, and clustered with germplasm of

distinct geographical collection. The germplasm grouped in

the cluster II were much more resolved and had twice the

genetic distance that of cluster I. This may be because of

the samples in the cluster II were more diverse and col-

lected from distinct geographical area. The overall den-

drogram of RAPD analysis resulted in random clustering of

germplasm and not in according to geographical area of

collection. Among all, JCI20 showed minimum GS with

the rest and found to be the most diverged germplasm

among characterized.

The dendrogram constructed using binary data of AFLP

showed good correlation with the dendrogram of RAPD

(Figs. 2, 3). As in RAPD dendrogram germplasm collected

from Gujarat (JCI02, 03, 06, 07, 08, 09) and Orissa (JCI01,

10) grouped together in cluster I of AFLP dendrogram. In

comparison the germplasm in major clusters of the

dendrograms, both in RAPD and AFLP observed to be the

same; however, sub clustering of germplasm showed minor

variations. AFLP dendrogram also showed no correlation

in clustering pattern to the geographical area of collection.

The overall comparative analysis of both the dendro-

gram RAPD and AFLP, the germplasm were placed in their

respective clusters with minor changes. However, AFLP

resulted in better resolution and superior marker poly-

morphism since it gave better mean PP among any two

germplasm compared and resulted with better resolved

dendrogram. As reported by Tatikonda et al. [11], the

present analysis also has revealed the germplasm belongs

to Uttar Pradesh, Madhya Pradesh and Gujarat clustered in

a same major cluster but separated by the germplasm

belongs to different geographical areas. The random

grouping of the germplasm belongs to distinct geographical

areas in the clustering of the dendrogram observed in the

present study was in agreement with earlier studies. [10].

The overall results obtained in this study showed good

congruence with RAPD and AFLP each other and imply

the competent use of the techniques for diversity study in J.

curcas. Similar results have been reported in apple [37, 38],

melon [28], accessions of Sorghum bicolor [29], blackgram

[39] and Saxifraga cernua [40] and the diversity found to

be more than the diversity observed in J. curcas. The

present study provided the back ground of genetic diversity

of J. curcas and the consistency of the result was supported

with bootstrap analysis in both RAPD and AFLP dendro-

grams. In both the dendrograms the formation of the two

major cluster may be due to that two distant germplasm

were introduced in India and were may subsequently

spread through out the country and the random clustering

resulted which was not in accordance with geographical

region may be due to antropogenic activity in distribution

of the species as earlier studies stated [10, 12]. The overall

analysis showed narrow diversity among the germplasm of

J. curcas in India.

The present study based on comparative analysis of both

RAPD and AFLP fingerprinting concludes the low genetic

diversity of J. curcas in India and will provide pavement for

further intra-population studies on narrow geographical

areas to understand the population genetic structure, phy-

logeography and molecular ecological studies needed for

developing the species as a potential crop. The germplasm

characterized and markers generated by RAPD and AFLP in

the present study can be employed efficiently in breeding

programmes for genetic improvement of the species through

marker assisted selection and QTL analysis, for further

genetic resource management and help in making the

J. curcas as potential crop with superior agronomical traits.

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

support.

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123

Molecular characterization and genetic diversity analysis of Jatropha curcas L. in India using RAPD and AFLP analysisAbstractIntroductionMaterials and methodsGenomic DNA extractionRAPD analysisAFLP analysisData analysis

ResultRAPD analysisAFLP analysis

DiscussionAcknowledgmentsReferences

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