Mol Biol Rep (2010) 37:22492257

DOI 10.1007/s11033-009-9712-2

Molecular characterization and genetic diversity analysis

of 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 Euphorbiaceae, 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 petrodiesel. Very few attempts were made to understand the
extent of genetic diversity that exists in J. curcas. Therefore, 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 germplasm JCI20 found to be the most diverged one. The
dendrogram analysis of RAPD and AFLP data showed
good congruence, but better resolution and more polymorphism 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

Electronic supplementary material The online version of this

article (doi:10.1007/s11033-009-9712-2) contains supplementary
material, 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

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-population studies on narrow geographical areas, to understand
the population genetic structure, phylogeography, molecular 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 renewable energy source [1, 2] and offer prospects by increasing
energy supplies in a self reliant way in developing countries 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 conventional 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

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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 characterization 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 species 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, population and/or germplasm of narrow geographical areas
only [7, 10, 11].
Therefore, the present investigation was undertaken to
characterize the germplasm collected from distinct geographical 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 germplasm (Table 1, Fig. 1) established in Central Salt and
Marine Chemicals Research Institute, Bhavnagar, Gujarat,
India, experimental field (21750 N, 72140 E). About 0.1 g
of 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,

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Mol Biol Rep (2010) 37:22492257

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 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 according 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 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 template, 1 unit Taq
DNA polymerase (Sigma, USA). Amplification was performed in programmed thermal cycler (Master cycle epgradient 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 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 37C
for 2 h and digested aliquot was ligated to EcoRI and MseI
specific adaptors at 20C for 90 min. The ligated DNA was
preamplified using EcoRI and MseI with one selective
nucleotide at the 30 end primer each. The preamplified
product 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. PCR
was performed using 65C as the initial annealing temperature 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 temperature. To the PCR product equal amount of formamide dye

Mol Biol Rep (2010) 37:22492257

Table 1 Details of the
geographic locations of J.
curcas gerplasm used in the
study

2251

Serial
number

Germplasm
code

State provenance
of collection (India)

Latitude

Longitude

JCI01

Orissa

218228N

8.580 8.680 E

JCI02

Gujarat

22, 74.38N

75, 57.846

JCI03

Gujarat

21560 , 18.45

72100 , 43.84

JCI04

Uttar Pradesh

26.45N

83.23E

JCI05

Uttar Pradesh

25.57N

81.50

JCI06

Gujarat

24, 16.35

72, 44.93

JCI07

Gujarat

21.50, 17.53

72.10, 44.96

JCI08

Gujarat

22, 17.350

75, 57.420

Gujarat

72, 46.930

9
10

JCI09
JCI10

Orissa

24, 17.43
0

84, 56.820

9, 25.44

11
12

JCI11
JCI12

Orissa
Orissa

19, 23.36
19, 27.420

84, 56.420
85, 2.080

13

JCI13

Arunachal Pradesh

270000

934200

14

JCI14

Madhya Pradesh

22.59, 38.1

75.89, 83.4

15

JCI15

Assam

267300

940100

16

JCI16

Andhra Pradesh

18.3N

83.3E

17

JCI17

Bihar

25.30N

85.14E

18

JCI18

Tamil Nadu

11.190 , 141

76.560 , 165

19

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

17350
18250 N

76420
77390 E

24

JCI24

Maharashtra

20420 2260 N

7720 7760 E

JCI25

Tamil Nadu

26

JCI26

Haryana

28.27N

77.01E

27

JCI27

Kerala

11.050 , 538

76.370 , 529

28

JCI28

Andhra Pradesh

17420 N

83240 E

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.

11.04 , 327

76.490 , 702

25

(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.

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 similarity (GS) was calculated using Jaccards coefficient of
similarity [16] with the help of NTSYS-pc package

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 technologies 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

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2252
Table 2 List of RAPD primers,
number of amplified products
and percentage of
polymorphism obtained by
analyzing 28 Indian germplasm
of J. curcas L

Mol Biol Rep (2010) 37:22492257

Primer

Sequence of
RAPD
primer 50 -30

Number of
polymorphic
markers

IDT E 5

TCAGGGAGGT

10.00

4.00

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
OPL5

AAGCGGCCTC
ACGCAGGCAC

6.00
1.00

10.00
9.00

37.50
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
OPP6

TCGGCACGCA
GTGGGCTGAC

4.00
4.00

2.00
2.00

66.67
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

Percentage of
polymorphism
71.43

OPQ15

GGGTAACGTG

4.00

2.00

66.67

OPQ2

TCTGTCGGTC

3.00

4.00

42.86

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 resolution 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

123

Number of
nonpolymorphic
markers

markers scored between JCI01 and JCI02 only one polymorphic 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; Gujarat (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),

Mol Biol Rep (2010) 37:22492257

2253

Fig. 1 Map displaying the

location of J. curcas germplasm
collected in India

Fig. 2 Dendrogram generated

using UPGMA for 28
germplasm of J. curcas based
on RAPD data obtained by 29
primer combinations. The
values on the nodes of the
cluster indicate the bootstrap
values and the scale represents
Jaccards similarity coefficient
values

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

Bihar (JCI17) and Jharkhand (JCI20). Among the germplasm 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 polymorphic. 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. Overall average PP given by primers was 61.24. The extent of

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2254
Table 3 List of AFLP selective
primer combination, number of
amplified products and
percentage of polymorphism
obtained by analyzing 28 Indian
germplasm of J. curcas L

Mol Biol Rep (2010) 37:22492257

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)
P18(MCAC/EACA)

27.00
14.00

22.00
20.00

55.10
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

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. Minimum 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), Karnataka (JCI23), Maharashtra (JCI24), Haryana (JCI26), Kerala (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

123

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]. Advances 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

Mol Biol Rep (2010) 37:22492257

2255

Fig. 3 Dendrogram generated

using UPGMA for 28
germplasm of J. curcas based
on AFLP data obtained by 18
primer combinations. The
values on the nodes of the
cluster indicate the bootstrap
values and the scale represents
Jaccards similarity coefficient
values

relatively small metacentric and submetacentric chromosomes whose size range from 1.71 to 1.24 lm [30]. Phylogenetic 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 intraspecific hybridization [12].
There were very few studies carried to understand the
diversity using various marker systems in J. curcas. Sujatha et al. [31] studied the extent of genetic diversity among
toxic and non-toxic varieties using RAPD and the percentage 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] characterized 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 amplification of minisatellite DNA (DAMD) primers. Novel
microsatellites isolated from J. curcas and were characterized 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 nontoxic 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 characterized 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 distinct geographical area showed good correlation in genetic
relatedness and clustering; however, AFLP showed superior marker polymorphism better resolution in the dendrogram analysis. PP in the present study showed 50.70
and 60.95% by RAPD and AFLP, respectively. The superior 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 introductions of the species and further distribution of the race
through vegetative propagation and by anthropogenic
activity as viewed in the earlier studies [7, 10].

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Although the germplasm were not clustered in accordance 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 consistency 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. Similarly 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 correlation in their clustering pattern was observed with geographical 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 collected from distinct geographical area. The overall dendrogram 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 dendrogram RAPD and AFLP, the germplasm were placed in their
respective clusters with minor changes. However, AFLP
resulted in better resolution and superior marker polymorphism 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

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Mol Biol Rep (2010) 37:22492257

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 dendrograms. 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, phylogeography 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 Scientific
and Industrial Research (CSIR), New Delhi, India for financial
support.

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