Mol Biol Rep (2010) 37:37853793

DOI 10.1007/s11033-010-0033-2

Isolation of novel microsatellites using FIASCO by dual probe

enrichment from Jatropha curcas L. and study on genetic
equilibrium and diversity of Indian population revealed
by isolated microsatellites
Pamidimarri D. V. N. Sudheer Hifzur Rahman
Shaik G. Mastan Muppala P. Reddy

Received: 16 October 2009 / Accepted: 24 February 2010 / Published online: 11 March 2010
Springer Science+Business Media B.V. 2010

Abstract Jatropha curcas L. belongs to family Euphorbiaceae, native to South 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
isolate novel microsatellite markers and assessment of the
extent of genetic equilibrium and diversity that exists in
J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic
equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India
using isolated microsatellites. The overall efficiency of the
enrichment of microsatellite by dual probe in the present
study found to be 54% and among the sequences obtained

Electronic supplementary material The online version of this

article (doi:10.1007/s11033-010-0033-2) contains supplementary
material, which is available to authorized users.
P. D. V. N. Sudheer  H. Rahman  S. G. Mastan 
M. P. Reddy (&)
Discipline of Wasteland Research, Central Salt and Marine
Chemicals Research Institute (CSIR), Bhavnagar,
Gujarat 364002, India
e-mail: reddymuppalareddy@gmail.com
P. D. V. N. Sudheer
e-mail: sudheer.vsc@gmail.com
Present Address:
P. D. V. N. Sudheer
Shree M. & N. Virani Science College,
Saurashtra University, Rajkot, Gujarat 360005, India
Present Address:
M. P. Reddy
Plant Stress Genomics and Technology Center,
King Abdullah University of Science and Technology,
Thuwal 23955-6900, Kingdom of Saudi Arabia

the percentage of sequences having suitable flanking

regions for the primer designing was found to be 89.58%.
The mean co-efficient of genetic similarity (CGS) was
found to be 0.97. The overall diversity obtained by
microsatellites was found to be low in comparison with the
diversity reported by multilocus markers systems observed
in earlier studies; however, the good allele polymorphism
was observed. The overall dendrogram of microsatellite
analysis resulted in random clustering of germplasm and
not in accordance to geographical area of collection. The
present study, diversity analysis using microsatellite
markers concludes the low genetic diversity and genetic
disequlibrium 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. The germplasm characterized, and the microsatellite
markers isolated and characterized in the present study can
be employed efficiently in breeding programs 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.
Keywords Biodiesel  Genetic diversity  J. curcas 
Microsatellites

Introduction
The genus Jatropha (Euphorbiaceae) includes 172 species
and is prominent in the flora of Africa and Asia. Among
these Jatropha curcas L. is a perennial shrub native to
South America, now grown in Asia and Africa [1]. J.
curcas has attained a significant importance as a potential

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biodiesel source because of its oil-rich seeds. Easy adaptation to different kind of marginal lands, drought endurance, avoidance by animals and its short interval time to
give first yield make this species more attractive for cultivation as a biodiesel plant. However, the crop is characterized by variable and unpredictable yield for the reasons
that have not been identified [2] 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.
There were few studies done in characterization of
natural diversity of J. curcas germplasm with various
multilocus markers systems and reports resulted in narrow
diversity limiting the species for various genetic improvement programs [1, 37]. So, it is necessary to employ more
powerful DNA markers to clarify the system. Generation of
novel molecular markers like microsatellites will provide
better tools to assess the amount and distribution of
molecular diversity, and for population genetic studies and
the isolated microsatellite markers will have better applications than multilocus markers systems.
In the last few years microsatellites have become one of
the most popular molecular marker used with applications in
different fields. As compared to multilocus markers like
RAPD and AFLP, microsatellites have advantages like locus
specificity, codominant nature, high reproducibility and
substantial size polymorphism [8]. Microsatellites or simple
sequence repeats (SSRs) are tandem repeating motifs of 16
bases found in all prokaryotic and eukaryotic genomes. In
addition to being highly variable, microsatellites are also
densely distributed throughout eukaryotic genomes, making
them as preferred marker for very-high-resolution genetic
mapping [9, 10]. Having huge applications the major draw
back with these microsatellite markers is they need to be
isolated de novo from most species being examined for the
first time. However, a numbers of protocols were designed to
obtain microsatellite markers. Among the enrichment techniques fast isolation by AFLP of sequences containing
repeats (FIASCO) is in principle; is readily applicable for the
labs where the AFLP is already standardized and also works
in a very cost efficient way [11]. In our previous study we
attempted to isolate the SSR from J. curcas using FIASCO
method; however, the efficiency obtained is less and many of
sequences were found not having sufficient flanking region
for primer designing [12].
The present study was under taken to isolate the more
number of markers by overcoming the previous discussed
problems and to study the diversity among diverse germplasm of J. curcas using the isolated novel microsatellites
combined with previously reported markers. The major
modification adopted in the enrichment in present study
was (1) partial digestion of genomic DNA (2) enrichment

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of the microsatellite fragment using two 50 biotin-labeled

oligonucleotide probes having GT and CT repeats.

Materials and methods

Genomic DNA extraction
Genomic DNA was extracted using CTAB protocol as
described by Sudheer et al. [13] from the 44 germplasm
collected from distinct geographical areas in India
(Table 1) established in Central Salt and Marine Chemicals
Research Institute, Bhavnagar, Gujarat, India, experimental
field (21750 N, 72140 E). 0.1 g of leaf tissue was ground
in liquid nitrogen and taken into a 2 mL microcentrifuge
tube. To the ground sample 0.5 mL of extraction buffer
(2% CTAB, 100 mM TrisHCl, 3.5 M NaCl, 20 mM
EDTA, 0.2 M b-Mercaptoethanol, 2% PVP, pH 8.0.) was
added and incubated at 65C for 90 min. The above
sample was extracted with equal volume of chloroform:
isoamyl alcohol (24:1) and supernatant was transferred
into a new tube. The 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.
Construction of SSR-enriched genomic libraries
FIASCO protocol described by Zane et al. [11] and minor
modifications done in our previous study [12] was further
modified to achieve better enrichment efficiency and to get
sufficient flanking region for primer designing. Genomic
DNA was partially digested using MseI/EcoRI (AFLP
analysis kit, Invitrogen, USA) and resulted fragments were
ligated to the MseI and EcoRI adaptors provided with the
kit. Amplification of adaptor-ligated fragments was performed using MseI and EcoRI adaptor primers without a 30
selective nucleotide. Fragments containing SSR sequences
were selected by hybridization with two biotinylated
[50 biotin(CT)1530 , 50 biotin(GT)1530 ] oligonucleotides. The
product was hybridized to 50 biotin-labelled oligonucleotide probe after denaturation. The DNA molecules bound to
the biotin-labelled probes were subsequently isolated by
binding to Streptavidin MagneSphere Paramagnetic
Particles (Promega). After rinsing the beads twice each
with washing buffers [TEN100 (10 mM TrisHCl, 1 mM
EDTA, 100 mM NaCl) pH 7.5; 19 SSC, 0.1% SDS], the
target DNA fragments were recovered by alkaline denaturation as described by Zane et al. [11]. The resultant
fragments were then amplified by polymerase chain reaction (PCR) using MseI and EcoRI primers without a 30

Mol Biol Rep (2010) 37:37853793

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

3787

Serial
number

Germplasm
code

State provenance
of collection (India)

Latitude

Longitude

JCI01

Orissa

218228 N

8.580 8.680 E

JCI02

Gujarat

22, 74.38 N

75, 57.846

JCI03

Gujarat

21560 , 18.45

72100 , 43.84

JCI04

Uttar Pradesh

26.45 N

83.23 E

JCI05

Uttar Pradesh

25.57 N

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

18.3 N

83.3 E

17

JCI17

Bihar

25.30 N

85.14 E

18

JCI18

Tamil nadu

11.190 , 141

76.560 , 165

19

JCI19

Chattishgadh

21.13 N

81.41 E

20

JCI20

Jharkhand

23.19 N

85.27 E

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

25

JCI25

Tamil Nadu

11.04 , 327

76.490 , 702

26
27

JCI26
JCI27

Haryana
Kerala

28.27 N
11.050 , 538

77.01 E
76.370 , 529

28

JCI28

Andhra Pradesh

17420 N

83240 E

29

JCI29

Gujarat

21.50, 17.53

30
31
32

JCI30
JCI31
JCI32

Karnataka
Karnataka
Gujarat

72.10, 44.96

76420 77390 E

76420 77390 E

1735 1825 N
1735 1825 N
0

22, 17.35
0

75, 57.420

33

JCI33

Andhra Pradesh

1742 N

83240 E

34

JCI34

Orissa

19, 27.420

85, 2.080

77.040

35

JCI35

Maharashtra

20 42

36

JCI36

Gujarat

21.50, 17.53

72.10, 44.96

37

JCI37

Gujarat

21.50, 17.53

72.10, 44.96

38

JCI38

Tamil Nadu

11.190 , 141

76.560 , 165

39

JCI39

Gujarat

24, 16.35

72, 44.93

40

JCI40

Rajasthan

24 42 03

73 38 13

41

JCI41

Haryana

28.27 N

77.01 E

42
43

JCI42
JCI43

Gujarat
Gujarat

24, 16.35
22, 17.350

72, 44.93
75, 57.420

44

JCI44

Andhra Pradesh

18.3 N

83.3 E

selective nucleotide for 12 cycles and the product was

purified with GenElute PCR Clean-Up Kit (Sigma). The
purified PCR products were cloned into T-vector (pTZ57R/

T; Fermentos, USA) and transformed into competent

Escherichia coli DH5a cells. The putative clone selection
was carried out based on blue/white selection and checked

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for insert by colony PCR using M13 universal primers. The

recombinant plasmids were extracted using GenElute
Plasmid Miniprep Kit (Sigma, USA). The sequences were
obtained using ABI PRISM 3100 Genetic Analyser
(Applied Biosystems) with M13 universal primers (IDT,
USA).
SSR analysis
Primers were designed to the flaking region of the isolated
microsatellite markers using NetPrimer software. PCR was
conducted for each microsatellite with designed primers
among the diverse germplasm to characterize the marker
polymorphism and genetic diversity among the germplasm.
Each PCR was performed in a volume of 20 lL containing
0.5 U Taq DNA polymerase (Sigma), 19 PCR buffer (mM
TrisHCl (pH 9.0), 50 mM KCl, 0.1 Triton X-100),
0.2 mM dNTPs mix, 2 lM of each primer set, 2.8 mM
MgCl2 and about 20 ng template DNA. Amplification was
performed in programmed thermal cycler (Master cycle
epgradient S, Eppendorf, Germany) programmed for an
initial denaturation at 94C for 3 min, 35 cycles of denaturation at 94C for 15 s, primer annealing (Table 2, supplementary material) for 20 s, extension at 72C for 30 s
and final extension at 72C for 4 min. Amplified products
were resolved in 8% denaturing long length polyacrylamide gel and visualized by silver staining. 20 bp DNA
ladder (Fermentos, USA) was used to define allele sizes.
Data analysis
Estimates of similarity were based on Nei and Lis [14]
definition as follows Sij = 2a/(2a ? b ? c), where Sij is
the similarity between two individuals, i and j, a is the
number of bands present both in i and j, b is the number of
bands present in i and absent in j, and c is the number of
bands absent in i and present in j. The similarity matrix was
then analysed using the clustering method UPGMA
(unweighted pair group method) [15] using the NTSYS 2.1
software [16]. The dendrogram was created with the tree
program of NTSYS, goodness of fit of the clustering to the
data was calculated using bootstrapping [17] across the loci
taking 1000 replicates and the score above 50 was entered
in the dendrogram (Fig. 3).

Results
The microsatellite library was constructed using the
enrichment procedure with two major modifications (1)
Digesting the genomic DNA partially rather than completely since in our previous study majority of the
sequences (20%) found lack of sufficient flanking regions

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[12], (2) Using two biotinylated oligos simultaneously

during enrichment. From the library constructed, out of 89
clones sequenced 48 sequences found to be containing
sequence repeats. Among the obtained sequence repeats,
43 sequences found to be having sufficient flanking regions
for designing the primers. The overall efficiency of the
enrichment found to be 54% and among the sequences
obtained, the percentage of sequences having proper
flanking regions found to be 89.58%. Among the 43
sequences 16 found to be perfect repeats and 27 found to be
imperfect repeats. Out of 43 sequences checked 32 loci
were amplified with expected size.
Total 49 SSRs were used in the present study to characterize the genetic diversity analysis of 44 diverse germplasm acquired form distinct geographical regions from
India (Fig. 1). Among the SSRs studied 36 loci were isolated in the present investigation, 12 microsatellites were
taken form our previous study and 5 loci were taken from
the study reported by Sun et al. [7]. At least one allele from
each locus with approximate expected size was able to
amplify in all 44 germplasm of J. curcas (Fig. 2; Table 2,
supplementary material).
Among 49 loci studied 2 loci resulted with monomorphic loci without polymorphic allele. Among the 47
polymorphic loci maximum number of alleles (11) were
obtained with locus JCDS24 followed by JCPS20 (9
alleles) Minimum number of alleles (2) were obtained from
13 loci (Table 2, supplementary material). From 47 polymorphic loci total of 172 alleles were observed and used
for the further diversity analysis and dendrogram analysis.
The mean number of alleles per locus in the present study
was found to be 3.51 alleles. The observed allele size
approximately ranged from 70 to 520 bp. The mean coefficient of genetic similarity (CGS) was found to be 0.97
(Table 3, supplementary material). The majority of the loci
showed low P-values due to the large variations in
expected to observed heterozygosities which show the
allele disturbances in population (Table 2, supplementary
material).
The dendrogram generated using the genetic similarity
showed a major cluster and five minor clusters. The major
cluster included with germplasm JCI15, JCI26, JCI41,
JCI17, JCI19, JCI16, JCI28, JCI33, JCI18, JCI38, JCI44,
JCI25, JCI27, JCI22, JCI40, JCI23, JCI30, JCI31 and it is
included with germplasm collected from the distinct state
provenances (Uttar Pradesh, Gujarat, Assam, Haryana,
Bihar, Chattishgadh, Andhra Pradesh, Tamilnadu, Kerala,
Rajasthan, Karnataka). The minor cluster I was formed
with the germplasm belongs to Gujarat (JCI03), Arunachal
Pradesh (JCI13) and Maharashtra (JCI 24 and JCI35).
Minor cluster II was formed only with germplasm belongs
to Gujarat (JCI06, JCI09, JCI42 and JCI39). Like as, minor
cluster V also formed with the germplasm (JCI01, JCI11,

Mol Biol Rep (2010) 37:37853793

3789

Fig. 1 Map displaying the

location of J. curcas germplasm
collected in India used in this
study

Fig. 2 Allelic polymorphism

among 44 accessions by
microsatellite marker JCPS20

Fig. 3 Dendrogram generated

using UPGMA for 44
germplasm of J. curcas based
on allele polymorphism data
obtained by 49 microsatellite
loci. The values on the nodes of
the cluster indicate the bootstrap
values (only values of above 50
are given) and the scale
represents Jaccards similarity
coefficient values

JCI12 and JCI34) belongs to single state provenance

(Orissa). The majority of the minor clusters formed by the
germplasm belong to single state provenances; however the
incorporation of the germplasm belongs to diverse

geographical areas was observed in the minor clusters I, III,

IV. Though the sample belongs to near geographical areas
clustered together, the overall pattern of the clustering was
not in accordance with the geographical areas of collection

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3790

and many clusters were represented with low bootstrap

values (Fig. 3).

Discussion
Many studies were carried out to analyze the diversity
within and among the population of plant species using
allozymes [18, 19]. The limitations with these techniques
were low number of markers and pseudo variations [19
24]. 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 marker systems microsatellites
were preferred markers to study the diversity and population genetic studies because of the markers were technically simple, high polymorphism and the variations
accumulate in accordance of the species evolution [11].
J. curcas, though having the huge importance as a bioenergy source the past molecular analysis with multilocus
marker systems like RAPD, ISSR, AFLP reported to be
giving limited number of polymorphic markers which is
limiting the efforts to improve the species through the
markers assisted breeding and selection procedures. From
identifying relatives to inferring demographic parameters,
microsatellites are rapidly replacing the multilocus markers
in most applications in population biology and molecular
genetics studies [2527] because of their advantages like
locus specificity, co-dominant nature, high reproducibility
and substantial size polymorphism [8]. Soon after their first
description [2830] SSRs were being widely employed in
many fields because of their high variability which made
them very powerful markers. In addition to being highly
variable, microsatellites are also densely distributed
throughout eukaryotic genomes, making them as preferred
marker for very high resolution genetic mapping, forensic
DNA studies, to population genetics and conservation
management of biological resources for genome mapping,
in hybrid analysis, MAS (Marker Assisted Selection) and
QTL (Quantitative Tract Loci) analysis for economically
important tract in many crop species [9, 10, 27, 31, 32].
The major drawback of microsatellites is that they need to
be isolated de novo from most species being examined for
the first time. So, isolation of the novel microsatellite
markers from J. curcas will help the molecular diversity
and phylogenetic analysis of the species population and
also the markers can be exploited for improvement of the
species through marker assisted breeding programs.
The task of microsatellite isolation traditionally was
quite involving in terms of effort and time because it
consists of screening genomic libraries with appropriate
probes and sequencing the positive clones. The number of
positive clones (containing microsatellites) that can be

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obtained by means of this traditional method usually ranges

from 12% to less than 0.04% [33]. A number of new
protocols overcoming these limitation were evolved like
the enrichment of microsatellites by hybridizing microsatellite containing oligo with biotin tagged that are further
captured by magnetic beads containing streptavidin or
fixed on nitrate filter became more popular [34] and has
been successfully applied to a number of plant genomes
[3436]. Among the enrichment techniques fast isolation
by AFLP of sequences containing repeats (FIASCO) is in
principle is readily applicable for the labs where the AFLP
is already standardized and works in a very cost efficient
way [5, 11, 37] and was successfully applied in J. curcas
[7, 12], but the efficiency was found to be less. The two
major problems faced in our previous study was less representative number of repeats containing fragment in the
enriched library and lack of flanking regions sufficient for
the designing primer.
To over come the problems two major modification
adopted in the enrichment in the present study (1) partial
digestion of genomic DNA (2) enrichment of the microsatellite fragment using two 50 biotin-labeled oligonucleotide probes having GT and CT repeats found to be resulted
better efficiency of the protocol. In the present study using
dual probe enrichment was found to be 53.93% where as
the earlier enrichment efficiency found to be 38.80%,
which concludes using dual probe in the enrichment will
result in better enrichment. Second major problem was
over come by partial digestion of the genomic DNA rather
than complete digestion, reduced the lack of flanking
regions from 20 to 10.41%. This is due to the reason that
the flanking region getting lost due to the complete
digestion will be retained when the digestion was partially
done and these sequences will subsequently get enriched.
J. curcas is a small genome species having the 2C
genome of 0.85 pg with 38.7% of average GC base composition. The karyotype of J. curcas is made up of 22
relatively small metacentric and submetacentric chromosomes whose size range from 1.71 to 1.24 lm [38]. There
were very few studies carried to understand the diversity
using various marker systems in J. curcas. Sujatha et al.
[39] 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. Senthil et al. [40] studied the diversity
among eight Jatropha species and three J. curcas accessions using ISSR markers and found 98.14% polymorphism across the species which are in accordance with our
previous results based on RAPD, AFLP and nrDNA ITS
analysis [1, 41].
In another study Sudheer et al. [37] 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-

Mol Biol Rep (2010) 37:37853793

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, whose application span over the selective cultivation of the specific
variety with better economical benefits [3]. In our previous
study the characterization in a population of J. curcas using
12 novel microsatellites showed significant deviation from
HardyWeinberg equilibrium [12]. 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 [5] and the similar observation was reported [3, 6]. Tatikonda et al. [6] studied the
diversity among the germplasm collected from six states
and the results were in agreement with our previous studies
[5]. Apart from the markers studies in Jatropha the efforts
were also made to isolate some gene cDNAs like translationally controlled tumor protein (TCTP) and phospholipase D for the characterization of these genes and their
influence in the physiology of the plant and environmental
stress which may help in improvement of the species using
modern biotechnology techniques [42, 43].
Genetic diversity analyses studies in J. curcas were
limited to some varieties and group of populations and/or
germplasm of narrow geographical area only [1, 3, 5, 6]. In
our previous study 28 germplasm of J. curcas were studied
and found to be having narrow genetic diversity which
limits the application in breeding programs. Characterization of germplasm and deducing the genetic equilibrium
and diversity using SSR which showed relatively high
polymorphism [12] will provide the 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 and in QTL
analysis.
In the present study the 44 germplasm acquired from
distinct geographical areas (Fig. 2, Table 1) studied for
their genetic equilibrium and diversity using novel microsatellites isolated in the present study as well as in previous
reports [7, 12]. The major correlations obtained were in
agreement with previous studies. The overall diversity
obtained by microsatellites found to be low in comparison
with the diversity reported by multilocus markers systems
including our recent studies [5] which in agreement with
results of Basha et al. [4]; this may be the reason that the
diversity obtained is using multilocus markers is representative of the overall genome; whereas, the diversity
obtained by SSRs is based on single locus polymorphism
and the analyzed loci are limited. In the present study the
diversity obtained among the narrow geographical distances was very much low in much cases it was found to be
nil e.g. germplasm from Orissa (JCI01, JCI11, JCI12 and
JCI34). The genetic equilibrium analysis showed only four

3791

markers are in accordance with HW equilibrium which

strongly supports the populations are not under the natural
distribution. Overall the narrow genetic diversity low
genetic equilibrium observed in case of J. curcas may be
because of few introductions of the species in the country
and further distribution of the race through vegetative
propagation and by anthropogenic activity because of its
valuable seeds as source of oil [2, 3, 5].
Although the overall clustering of the germplasm was
not in accordance to the geographical areas, but the
germplasm belongs to single state provenances were clustered together. The clustering patterns observed in the
constructed dendrogram using SSR data is in correlation
with our previous analysis done using multilocus marker
system [5]. As in the RAPD and AFLP dendrogram, in
present study the dendrogram the major cluster was much
more resolved and had twice the genetic distance that of
other minor clusters obtained. This may be because of the
samples in the major cluster were more diverse and
included with germplasm belongs to highly distinct state
provinces. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in
accordance with geographical area of collection. As
observed in our earlier study using RAPD and AFLP [5]
where the germplasm JCI20 showed minimum GS with the
rest of the germplasm found to be the same in the present
analysis and was separated from all the germplasm and
formed distinct clade in the dendrogram. As reported by
Tatikonda et al. [6], the present analysis also has revealed
that the germplasm belonging to Uttar Pradesh, Madhya
Pradesh and Gujarat clustered in a same major cluster but
separated by the germplasm belonging 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 [3, 5]. Low P-values, high
deviation from HW equilibrium and low bootstrap values
in the dendrogram represents the low phylogenetic or
geographical relations among the collected germplasm; this
is may be due to the anthropogenic activity in distribution
of the species rather than the natural distribution. This over
all analysis warrants for the genetic resource management
by allowing the natural dispersion of the race in at least
natural habitat and conservation of the diverse germplasm.
The present study provided the back ground of genetic
equilibrium and diversity of J. curcas and the genetic
relationships among the germplasm belongs to distinct
geographical regions. Though the genetic diversity
obtained in the present study was less than the previous
study using multilocus marker systems (RAPD, ISSR and
AFLP), good correlation was obtained in clustering patterns in the dendrograms and highly polymorphic microsatellite markers whose allele polymorphism was better

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appreciable were observed. In the present study the

diversity analysis using microsatellite markers concludes
the low genetic diversity and genetic disequlibrium of
J. curcas in India; high allele polymorphic microsatellite
loci were identified, and will provide pavement for further
intra-population studies on narrow geographical areas to
understand the population genetic structure, phylogeography and molecular ecological studies and for developing
the species as a potential crop. The germplasm characterized and the microsatellite markers isolated and characterized in the present study can be employed efficiently in
breeding programmes for genetic improvement of the
species through MAS and QTL analysis, for further genetic
resource management and help in making the J. curcas as
potential crop with superior agronomical traits.

Mol Biol Rep (2010) 37:37853793

11.
12.

13.

14.

15.

16.
Acknowledgments The authors wish to thank Council for Scientific
and Industrial Research (CSIR), New Delhi, India for financial support and for the Research Associate fellowship.

17.
18.

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