See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257372041

Evaluation of genetic diversity in turmeric

(Curcuma longa L.) using RAPD and ISSR
markers
Article in Industrial Crops and Products May 2012
DOI: 10.1016/j.indcrop.2011.12.022

CITATIONS

READS

31

308

3 authors:
Shikha Singh

Manoj Kumar Panda

Siksha O Anusandhan University

Siksha O Anusandhan University

27 PUBLICATIONS 92 CITATIONS

16 PUBLICATIONS 167 CITATIONS

SEE PROFILE

SEE PROFILE

Sanghamitra Nayak
Siksha O Anusandhan University
86 PUBLICATIONS 625 CITATIONS
SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Malaria (Plasmodium falcipatum and Plasmodiun vivax )diagnosis from urine samples of
human. View project
All in-text references underlined in blue are linked to publications on ResearchGate,
letting you access and read them immediately.

Available from: Shikha Singh

Retrieved on: 24 September 2016

Industrial Crops and Products 37 (2012) 284291

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Evaluation of genetic diversity in turmeric (Curcuma longa L.) using RAPD and
ISSR markers
Shikha Singh, Manoj Kumar Panda, Sanghamitra Nayak
Centre of Biotechnology, Siksha O Anusandhan University, Khandagiri, Bhubaneswar 751003, Orissa, India

a r t i c l e

i n f o

Article history:
Received 24 August 2011
Received in revised form
13 December 2011
Accepted 15 December 2011
Available online 14 January 2012
Keywords:
Turmeric
Genetic diversity
Agroclimatic zones
RAPD
ISSR
Molecular markers

a b s t r a c t
Turmeric (Curcuma longa L.) is an industrially important plant used for production of curcumin, oleoresin
and essential oil. In the present study we examined the genetic diversity among turmeric accessions from
10 different agro-climatic regions comprising 5 cultivars and 55 accessions. Two DNA-based molecular
marker techniques, viz., random amplied polymorphism DNA (RAPD) and inter simple sequence repeat
(ISSR) were used to assess the genetic diversity in turmeric genotypes. A total of 17 polymorphic primers
(11 RAPDs and 6 ISSRs) were used in this study. RAPD analysis of 60 genotypes yielded 94 fragments
of which 75 were polymorphic with an average of 6.83 polymorphic fragments per primer. Number of
amplied fragments with RAPD primers ranged from 3 to 13 with the size of amplicons ranging from
230 to 3000 bp in size. The polymorphism ranged from 45 to 100 with an average of 91.4%. The 6 ISSR
primers produced 66 bands across 60 genotypes of which 52 were polymorphic with an average of
8.6 polymorphic fragments per primer. The number of amplied bands varied from 1 to 14 with size of
amplicons ranging from 200 to 2000 bp. The percentage of polymorphism using ISSR primers ranged from
83 to 100 with an average of 95.4%. Neis dendrogram for 60 samples using both RAPD and ISSR markers
demonstrated an extent of 62% correlation between the genetic similarity and geographical location. The
result of Neis genetic diversity (H) generated from the POP gene analysis shows relatively low genetic
diversity in turmeric accessions of South eastern ghat (P7), Western undulating zone (P8) with 0.181 and
0.199 value whereas highest genetic diversity (0.257) has been observed in Western central table land
(P9). Knowledge on the genetic diversity of turmeric from different agro-climatic regions can be used
to future breeding programs for increased curcumin, oleoresin and essential oil production to meet the
ever-increasing demand of turmeric for industrial and pharmaceutical uses.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Turmeric (Curcuma longa L. Zingiberaceae), an industrially
important crop widely cultivated and highly exportable spice in
India, having unique chemical and physical properties and broadly
used for numerous industrial applications, such as manufacture
of canned beverages, baked products, dairy products, ice cream,
yogurt, yellow cakes, orange juice, biscuits, popcorn color, sweets,
cake icings, cereals, sauces, gelatins, and curry powders. Turmeric
is used as a food additive, preservative and coloring agent in Asian
countries (Antunes and Araujo, 2000; Ceclio-Filho et al., 2000).
In addition, turmeric has also a variety of pharmacological activities, with recent ndings which shows that curcumin, the yellow
color pigment of turmeric, is a powerful antioxidant, anti-parasitic,
antispasmodic and anti-inammatory compound, which may also
inhibit carcinogenesis (Araujo and Leon, 2001; Ravindran, 2007).

Corresponding author.
E-mail address: sanghamitran24@gmail.com (S. Nayak).
0926-6690/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2011.12.022

Indian enjoys monopoly in turmeric production and export.

Because of its ever-increasing demand in both food and pharmaceutical industries, there is pressing need to further increase the
productivity of turmeric. However, to obtain further increases in
productivity, information regarding the crops genetic diversity is
essential for breeding programs (Nass, 2001). Turmeric is a crosspollinated, triploid species (2n = 3x = 63), which can be vegetatively
propagated using its underground rhizomes (Sasikumar, 2005).
Since hybridization is ineffective in most cases, genetic improvement work on turmeric is mostly conned to germplasm selection
(Ravindran, 2007). Even though germplasm collections and characterization is prerequisite for genetic improvement of turmeric,
such studies are scarce and mostly restricted to the phenotypic
evaluation of accessions from different states of India including
Orissa, which is the second largest producer of turmeric in India
(Chaudhary et al., 2006). The use of phenotypic traits in germplasm
characterization is also limited due to the availability of small
number of descriptors. Characterization of promising turmeric cultivars/accession by morphological data and qualitative traits like
curcumin, oleoresin and essential oil content are not sufcient as

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

these characters often change under varying environmental conditions thus raising problem in proper identication and elimination
of synonyms.
Predominance of synonyms possesses problems in identication and characterization of germplasm. Many synonyms can be
removed by molecular characterization thus reducing the cost of
maintenance of redundants in clonal repositories. Due to resurgence of interest in the commercial production of different cultivars
of turmeric as new spice crops, it has become necessary to precisely characterize the genetic variations that exist in cultivars,
advanced selection and native population. This is one step toward
providing accurate genetic information for future breeding program for turmeric improvement. Molecular markers (RAPD, ISSR,
SSR, etc.) unlike morphological and biochemical markers are not
prone to environmental inuence and accurately characterize
the plants portraying the extent of genetic diversity among taxa
(Thimmappaiah et al., 2009; Cheng and Huang, 2009). Of the different molecular markers RAPD and ISSR has been widely used in the
last two decades in cultivar identication program (Ebrahimi et al.,
2009) and assessing genetic variations among different taxa at DNA
level because of their cost effectiveness and simple operation without requiring prior knowledge of species DNA sequences (Williams
et al., 1990) and can provide vital information for development
of genetic sampling, conservation and improvement strategies
(Chalmers et al., 1994). No report has yet been published so far
on agro-climatic zone based genetic characterization of existing
promising cultivars/accession of turmeric using molecular markers.
Available report on detection of genetic variation in 17 promising
cultivars of turmeric by (Nayak et al., 2006) necessitate more detail
work on genetic characterization of turmeric cultivars and accessions from 10 different agro-climatic regions of Orissa using RAPD
and ISSR markers.

Table 1
Details of the sampling areas of turmeric from different agroclimatic regions of India.
Agro-climatic zone (code)

Place of collection

Accession no.

North western plateau (P1)

Sundargarh
Deogarh
Redhakhol
Titlagarh
Jharsuguda
Patbil
Mayurbhanj
Ghasipura
Padiabeda
Anandpur
Palashpala
Keonjhar
Panikoili
Boudh
Bhadrak
Nilgiri
Jaleswar
Soro
Hatdihi
Khurda
Athagarh
Niali
Kandarpur
Rahama
Puri
Nayagarh
Ganjam
Draingabadi
G. udaigiri
Tikabali
Phulabani
Koraput
Rayagada
Parlakhemundi
Gajapati
Potangii (cv Surama)
Potangii (cv Roma)
Potangii (cv Ranga)
Pottangi (cv Rasmi)
Pottangi (cv Lakadong)
Nabarangpur
Bandaghati
Keonjhar
Malkangiri
Patbil
Bhawanipatna
Nuapada
Kalahandi
Kahdhamal
Balangir
Barapalli
Boudh
Sonepur
Sambalpur
Jharsuguda
Kuchinda
Angul
Dhenkanal
Cuttack
Jajpur

P1-1
P1-2
P1-3
P1-4
P1-5
P2-1
P2-2
P2-3
P2-4
P2-5
P2-6
P2-7
P3-1
P3-2
P3-3
P3-4
P3-5
P3-6
P3-7
P4-1
P4-2
P4-3
P4-4
P4-5
P4-6
P4-7
P4-8
P5-1
P5-2
P5-3
P5-4
P5-5
P5-6
P5-7
P5-8
P6-1
P6-2
P6-3
P6-4
P6-5
P6-6
P7-1
P7-2
P7-3
P7-4
P8-1
P8-2
P8-3
P8-4
P9-1
P9-2
P9-3
P9-4
P9-5
P9-6
P9-7
P10-1
P10-2
P10-3
P10-4

North central plateau (P2)

North eastern coastal plain

(P3)

East and south eastern

coastal plain (P4)

North eastern ghat (P5)

Eastern ghat highland (P6)

2. Materials and methods

2.1. Plant material and DNA extraction
South eastern ghat (P7)

The present investigation deals with 55 turmeric accessions

and ve cultivars collected from ten different agro-climatic
zones (P1P10) of India including ve cultivars collected from
High Altitude Research Station, Pottangi of Orissa University
of Agriculture and Technology, Koraput (Table 1). They were
maintained in the greenhouse of Centre of Biotechnology of
Siksha O Anusandhan University. Fresh leaf samples were
frozen in liquid nitrogen and preserved in 85 C freezer for
molecular analysis. Genomic DNA was isolated following the
protocol of Doyle and Doyle (1990) with little modication.
Fresh leaves (2 g) were ground in liquid nitrogen and taken
into 50 ml of centrifuge tube. To the ground sample 10 ml
of extraction buffer [4% cetyl trimethyl ammonium bromide
(CTAB), 100 mM TrisCl, 4 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 2% mercaptoethanol and 2% polyvinyl
pyrrolidone, pH 8] was added and incubated at 65 C for 1 h.
Then above sample was extracted with equal volume of chloroform:isoamyl alcohol (24:1), supernatant was treated with RNase
at 60 g ml1 and DNA was 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 more, and the DNA was precipitated with 80%
ethanol. The pellet was air dried and resuspended in 100 l
of TrisEDTA (TE Buffer). Puried total DNA was quantied in
0.8% agarose gel along with known amount of uncut lambda
DNA (Bangalore Genei Pvt. Ltd., Bangalore, India) as standard.
The sample DNA was diluted as 25 ng l1 for RAPD and ISSR
analysis.

285

Western undulating zone

(P8)

Western central table land

(P9)

Mid central (P10)

2.2. RAPD amplication

Eleven random decamer primers (Operon Tech, USA) from A, C,
D and N series (OPA04, 07, 08, 09, 18; OPC02, 05, 11; OPD08, 16, 18,
20; and OPN04, 06, 16,) were used for RAPD analysis. RAPD were
performed in a nal volume of 25 l containing 25 ng of template
DNA, 2.5 l of 10 assay buffer (100 mM TrisCl, pH 8.3, 500 mM
KCl, and 0.1% gelatin), 15 mM MgCl2 , 200 M of each dNTPs, 15 ng
of primer and 0.5 unit of Taq DNA polymerase (Bangalore Genei Pvt.
Ltd., Bangalore, India). The RAPD analysis was performed as per the
methodology described by (Williams et al., 1990) using Gene Amp

286

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

PCR System 9700 Applied Biosystem, USA. The rst cycle consisted
of denaturation of template DNA 94 C for 5 min, primer annealing
at 37 C for 1 min and primer extension at 72 C for 2 min. In the
next 42 cycles the period of denaturation was reduced to 1 min
at 92 C while the primer annealing and primer extension time
remained same as in the rst cycle. The last cycle consisted of only
primer extension at 72 C for 7 min. The reactions ended with an
indenite hold at 4 C. Amplied products were electrophoresed
in 1.5% agarose gels containing ethidium bromide 0.5 g ml1 in
TAE buffer (40 mM Tris base, 20 mM sodium acetate, 20 mM EDTA,
glacial acetic acid to pH 7.2) for 3 h at 60 V and documented using gel
documentation system (Bio Rad, California, USA). Each experiment
was repeated two times with each primer and those primers which
gave reproducible ngerprints (DNA bands) were only considered
for the data analysis.

Fig. 1. Result of gel electrophoresis of PCR products obtained by using (OP N06)
RAPD primer.

2.3. ISSR amplication

Six numbers of primers were used namely (GTGC)4 , (GACA)4 ,
(AGG)6 , (GA)9 T, T(GA)9 and (GTG)5 for ISSR analysis. These simple sequence repeats were synthesized and procured from Genei
(Bangalore Genei Pvt. Ltd., Bangalore, India). The ISSR analysis was
performed as per the methodology given by (Zeitkiewicz et al.,
1994). Each amplication reaction mixture of 25 l contained 25 ng
of template DNA, 2.5 l of 10 assay buffer (100 mM TrisCl pH
8.3, 0.5 M KCl and 0.01% gelatin), 1.5 mM MgCl2 , 200 m each of
dNTPs, 44 ng of primer and 0.5 U Taq DNA polymerase. The amplication was carried out in a thermal cycler. The rst cycle consisted
of denaturation of template DNA at 94 C for 5 min, primer annealing at specic temperature for particular primer for 1 min and
primer extension at 72 C for 2 min. In the subsequent 42 cycles
the period of denaturation was reduced to 1 min while the primer
annealing and primer extension time was maintained same as in
the rst cycle. The last cycle consisted of only primer extension
at 72 C for 7 min. Amplication products were electrophoresed
in 2% agarose gels containing ethidium bromide 0.5 g ml1 in
TAE buffer (40 mM Tris base, 20 mM sodium acetate, 20 mM EDTA,
glacial acetic acid to pH 7.2) for 3 h at 60 V. A total of 2.5 l loading
buffer (10 TAE, 50% glycerol, 0.25% bromophenol blue and 0.25%
xylene cyanol) was added to each reaction before electrophoresis.
After electrophoresis, gels were observed under UV-illuminator,
documented in Universal Hood II (Bio Rad, California, USA) and
photographed. Each experiment was repeated two times with each
primer and those primers which gave reproducible ngerprints
(DNA bands) were only considered for the data analysis.
2.4. Data analysis
The data was scored as 1 for the presence and 0 for the
absence of the band for each primer genotype combination for
RAPD and ISSR analysis. A dendrogram was constructed using the
unweighted pair group method with arithmetic average (UPGMA)
with the SAHN module of NTSYS-pc to show a phonetic representation of genetic relationships as revealed by the similarity
coefcient (Sneath and Sokal, 1973). Resolving power of the RAPD
and ISSR markers
were calculated as per Prevost and Wilkins

IB (IB, band informativeness = 1 [2 (0.5 P)]), P is
(1999), Rp =
the proportion of the species containing 
the band. A polymorphic
index (PIC) was calculated as PIC = 1
Pi2 , Pi is the band frequency of the ith allele (Smith et al., 1997). In the case of RAPDs
and ISSRs, the PIC was considered to be 1 p2 q2 , where p is band
frequency and q is no band frequency (Ghislain et al., 1999). Data
matrices of 2 different types of markers (RAPD and ISSR) as well
as combined markers were analyzed with POPGEN version 1.32
software for analyzing genetic diversity parameters like percentage
of polymorphic bands (PPB); observed number of alleles per locus

(Na), effective number of alleles per locus (Ne). Genetic diversity

measures (Ht, total gene diversity; Gst, coefcient of gene differentiation) were tested using (Neis 1973) gene diversity statistics.
Shannons information index (I) was also used to examine partitioning of genetic diversity within and among populations. Estimate of
gene ow (Nm) was calculated as Nm = 0.5(1 Gst)/Gst, where Gst is
the gene differentiation. Analysis of molecular variance (AMOVA)
was also used to obtain the variation component among individuals within populations and among populations. AMOVA input les
were created using AMOVA-PREP version 1.01 (Miller, 1998) and
the analyses were made using AMOVA version 1.5 (Excofer et al.,
1992).
3. Results
3.1. RAPD analysis
Total 30 RAPD primers were screened of which 11 primers
showed reliable banding patterns with high reproducibility and
clear band resolution. All DNA samples of 5 cultivars and 55
accessions produced distinct reproducible amplications with 11
selected RAPD primers. These 11 primers produced a total of 94
distinct amplication products ranging from 250 to 3000 bp. The
DNA proles as observed in RAPD are represented in (Fig. 1). The
number of scored bands per primer ranged from 5 (OPC02, OPN06)
to 13 (OPC05), with a mean number of 8.54 per primer (Table 2).
These two primers are also representatives of the lowest (2) for
OPC02 and the highest (13) for OPC05, number of polymorphic
bands. A total of 94 numbers of bands were amplied, of which 75
bands were found to be polymorphic and only 19 were monomorphic in nature. The average number of polymorphic markers across
the primers was 78.7% ranging from 40% produced by the primer
OPC02 to 100% obtained by other primers (OPC05, OPC11, OPN06
and OPN16) (Table 2).
The dendrogram constructed using the SAHN clustering separated the 60 samples into two major clusters one with 12 samples
and rest with samples as in Fig. 2. These two shared a common
node at 61% similarity level. The larger cluster was further subdivided into two subclusters of which one subcluster contains 13
samples being separated from rest all 35 subclusters sharing a common node at 62.5% similarity level. The second subcluster of 16
groups contained two small groups of 7 samples and the other
group of 9 samples sharing a node with them at approximately
63% similarity levels (Fig. 2). Resolving power was maximum (18.4)
for (OPC11) and minimum of (5.86) for the primer (OPN16). The
primer (OPC05) showed maximum primer index (5.46) (Table 2).
The percentage of polymorphic loci among the accessions of all
the 10 agro climate zones varied in the range of 51.06% in P8 to
72.34% in P9, with an average value of 75.53%. The samples of P9

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

287

Table 2
Details of banding pattern revealed through RAPD and ISSR primers.
Markers

Primer/primer
combination

Sequence of
the primers

Range of
amplicons in
bp

RAPD

OPA18
OPC02
OPC05
OPC11
OPD08
OPD16
OPD18
OPD20
OPN04
OPN06
OPN16

AGGTGACCGT
GTGAGGCGTC
GATGACCGCC
AAAGCTGCGG
GTGTGCCCCA
AGGGCGTAAG
GAGAGCCAAC
ACCCGGTCAC
GACCGACCCA
GAGACGCACA
AAGCGACCTG
Total

1200375
1350350
>3000375
1700250
2000650
2200450
1800550
2050400
1200500
1050250
1300250

10
5
13
12
8
11
6
10
6
5
8
94

8
2
13
12
5
6
3
9
4
5
8
75

ISSR

(GA)9 T
(GGA)4
(AGG)6
(CAA)5
(GACA)4
(GTG)5

1800250
1950200
1350250
2000475
1500300
1500250

11
14
12
12
8
9
66
160

Total
Grand total

Total
bands

Unique
bands

% of polymorphic
loci

Resolving
power (Rp)

Primer
index (PI)

2
3
0
0
3
5
3
1
2
1
0
20

0
0
0
0
0
0
0
0
0
0
0
0

100
60
100
100
100
45
100
100
100
100
100
91.4

12
8.9
7.8
18.4
16
16.46
8.4
18
12
7.3
5.86

4.8
0.74
5.46
4.29
0
2.82
2.52
1.8
0
1.95
3.71

11
14
10
8
8
1
52

0
0
2
4
0
8
14

0
0
0
0
0
0
0

100
100
83
100
100
88
95.4

0
8.7
19.03
15.4
9.8
16.9

0
4.49
2.34
3.29
2.8
0.495

126

34

Polymorphic Monomorphic
bands
bands

Table 3
Summary of genetic variations as revealed through RAPD markers among 10 populations of C. longa.
Population code
and size

Observed no. of
alleles (Na)
(mean SD)

P1
P2
P3
P4
P5
P6
P7
P8
P9
P10

1.5745
1.6489
1.7128
1.6809
1.7128
1.6702
1.5213
1.5106
1.7234
1.5319

Mean of all
populations

1.7553 0.4322

0.4971
0.4799
0.4549
0.4686
0.4549
0.4727
0.5022
0.5026
0.4497
0.5017

Effective no. of
alleles (Ne)
(mean SD)
1.4484
1.4796
1.5255
1.4917
1.5298
1.4953
1.3936
1.3915
1.5201
1.4000

0.4198
0.4005
0.3874
0.3948
0.3923
0.3939
0.4048
0.4105
0.3869
0.4032

1.5525 0.3791

Neis genetic
diversity (H)
(mean SD)

Total gene
diversity (Ht)
0.2468
0.2683
0.2944
0.2763
0.2955
0.2778
0.2207
0.2181
0.2927
0.2247

0.0488
0.0444
0.0407
0.0425
0.0412
0.0430
0.0472
0.0480
0.0398
0.0469

0.2468
0.2683
0.2944
0.2763
0.2955
0.2778
0.2207
0.2181
0.2927
0.2247

0.3089 0.0387

region have highest (1.7234 0.4497) observed number of alleles

(Na) and of P8 (1.5106 0.5026) has the lowest; the average being
1.755 0.432 (Table 3). The effective number of alleles (Ne) was
invariably less than Na values for each population showing a variation in the range of 1.40 (P10) to 1.52 (P5) with an average of
1.55. Neis gene diversity (H) and Shannons Index (I) were highest in populations collected from P5 zone (H = 0.295 0.203 and
I = 0.428 0.286) and lowest values were recorded for P8 (Table 3).
Therefore, among the groups of turmeric genotypes studied, P9
exhibited the greatest level of variability (PPB = 72.34%). Analysis of

No. of
polymorphic
loci

Percentage of
polymorphic loci
(PPB)

0.3135
0.2987
0.2850
0.2913
0.2861
0.2942
0.3113
0.3134
0.2805
0.3105

54
61
67
64
67
63
49
48
68
50

57.45
64.89
71.28
68.09
71.28
67.02
52.13
51.06
72.34
53.19

0.4485 0.2763

71

75.53

Shannons
information index
(I) (mean SD)

0.2209
0.2106
0.2018
0.2062
0.2030
0.2074
0.2172
0.2192
0.1994
0.2166

0.3554
0.3896
0.4276
0.4025
0.4287
0.4034
0.3196
0.3150
0.4269
0.3256

0.3089 0.1968

molecular variance (AMOVA) made using RAPD data showed that

42% of the total genetic variability can be accounted for the differences among the accessions of 10 agro-climate zone of Orissa. The
remaining 58% variations are due to variations among individuals
within the regions (Table 4).
3.2. ISSR analysis
Total 21 ISSR primers were screened and 6 primers amplifying more than six scorable bands were selected for further study.

Table 4
Analysis of molecular variance using RAPD, ISSR and combined markers.
Markers
RAPD
Among Pops.
Within Pops.
ISSR
Among Pops.
Within Pops.
Combined
Among Pops.
Within Pops.

df

SS

MS

Est. Var.

Value (%)

P-value

9.000
50.000

26.781
28.202

2.976
0.564

0.405
0.564

42
58

0.002

9.000
50.000

27.085
23.399

3.009
0.468

0.427
0.468

48
52

0.001

9.000
50.000

27.485
23.399

3.054
0.468

0.434
0.468

48
52

0.001

df, degree of freedom; SS, sum of square; MS, mean square; Est. Var., estimated variation.

288

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

Table 5
Summary of genetic variations as revealed through ISSR markers among 10 populations of C. longa.
Population code
and size

Observed no. of
alleles (Na)
(mean SD)

P1
P2
P3
P4
P5
P6
P7
P8
P9
P10

1.3485
1.3939
1.4545
1.6364
1.5606
1.5606
1.2879
1.4091
1.6061
0.5758

Mean of all
population

1.7879 0.4119

0.4801
0.4924
0.5017
0.4847
0.5001
0.5001
0.4562
0.4954
0.4924
0.4980

Effective no. of
alleles (Ne)
(mean SD)
1.2257
1.2712
1.3128
1.3299
1.3336
1.3217
1.2273
1.3061
1.3359
1.4182

0.3376
0.3691
0.3888
0.3304
0.3604
0.3491
0.3760
0.3910
0.3349
0.3887

1.4128 0.3605

Total gene
diversity (Ht)
0.1333
0.1558
0.1781
0.2050
0.1989
0.1953
0.1250
0.1723
0.2066
0.2386

0.0359
0412
0.0435
0.0322
0.0383
0.0357
0.0404
0.0451
0.0343
0.0446

0.2469 0.0335

Fig. 2. Dendrogram showing the clustering pattern among 60 samples of turmeric

as revealed through RAPD markers.

All DNA samples of 60 genotypes produced distinct reproducible

amplications with six selected ISSR markers with good reproducibility and resolution. The ISSR prole of all the samples is
represented in (Fig. 3). A total of 66 bands were amplied with six
primers, of which 52 were polymorphic and 14 monomorphic ones.
Maximum (14) amplication was observed in case of the primer
(GGA)4 and minimum (8) in case of (GACA)4 . Among these six
primers no unique band was detected for the primers tested so far.

Fig. 3. Result of gel electrophoresis of PCR products obtained by using (GACA)4 ISSR
primer.

Neis genetic
diversity (H)
(mean SD)
0.1333
0.1558
0.1781
0.2050
0.1989
0.1953
0.1250
0.1723
0.2066
0.2386

0.1895
0.2029
0.2084
0.1796
0.1956
0.1890
0.2010
0.2123
0.1852
0.2111

0.2469 0.1831

No. of
polymorphic
loci

Percentage of
polymorphic loci
(PPB)

0.2773
0.2930
0.2990
0.2610
0.2829
0.2761
0.2870
0.3053
0.2704
0.3042

23
26
30
42
37
37
19
27
40
38

34.85
39.39
45.45
63.64
56.06
56.06
28.79
40.91
60.61
57.58

0.3775 0.2517

52

78.79

Shannons
information index
(I) (mean SD)
0.1979
0.2288
0.2617
0.3158
0.2991
0.2961
0.1797
0.2499
0.3145
0.3476

Fig. 4. Dendrogram showing the clustering pattern among 60 samples of turmeric

as revealed through ISSR markers.

Resolving power was maximum (19.03) for (AGG)6 and minimum

(0) for the primer (GA)9 T. The primer (GGA)4 showed maximum
primer index (4.49) (Table 2) A dendrogram was constructed using
SAHN clustering through UPGMA from the ISSR data (Fig. 4). The
dendrogram separated the 60 samples into two major clusters, one
smaller with 5 samples and second larger with 55 samples (Fig. 4).
These two shared a common node at 55% similarity level. The larger
cluster was further subdivided into 2 subclusters containing only 1
sample in 1st subcluster and 53 samples in 2nd subcluster. These
2 subclusters shared a common node at 57.5% similarity level. The
2nd subcluster of 53 samples were further divided into 2 groups
of which the smaller group with 5 samples and the larger group
with 46 samples sharing a common node at 62.5% similarity level
(Fig. 4). Among the entire agro-climate zone studied on an average 78.79% of the loci were polymorphic; the minimum (28.79%)
and maximum (63.6%) polymorphism being detected in (P7) and
(P4) zone respectively. The mean observed number of alleles (Na)
of all population was 1.78. Highest observed number of alleles (Na)
and effective number of alleles (Ne) (1.63) and lowest (0.57) were
found in P4 and P10 populations respectively. The mean effective
number of alleles (Ne) for all the populations was 1.41 (Table 5).
Similarly, Neis gene diversity (H) and Shannons Information Index
(I) determined for P10 population were highest of all populations
(H = 0.238; I = 0.347) (Table 4). ISSR analysis revealed that the P9
population possesses relatively higher genetic diversity in comparison to the other 9 populations.

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

289

Table 6
Summary of genetic variations as revealed through combined markers among 10 populations of C. longa.
Population code
and size

Observed no. of
alleles (Na)
(mean SD)

P1
P2
P3
P4
P5
P6
P7
P8
P9
P10

0.5012
1.5437
1.6062
1.6625
1.6500
1.6250
1.4250
1.4688
1.6750
1.5500

Mean of all
population

1.7688 0.4230

Effective no. of
alleles (Ne)
(mean SD)

1.4812
0.4996
0.4901
0.4743
0.4785
0.4856
0.4959
0.5006
0.4698
0.4991

1.3566
1.3936
0.4008
1.4250
1.4489
1.4237
1.3250
1.3562
1.4441
1.4075

0.4022
0.4001
1.4377
0.3771
0.3905
0.3846
0.4005
0.4036
0.3764
0.3961

1.4949 0.3768

Total gene
diversity (Ht)
0.2000
0.2219
0.2464
0.2469
0.2557
0.2438
0.1812
0.1992
0.2571
0.2305

0.0464
0.0459
0.0449
0.0393
0.0420
0.0414
0.0463
0.0470
0.0391
0.0457

0.2833 0.0373

Out of the total variation, 48% of the variation was among the
accessions of different regions and the rest 52% was due to within
the region variability. Analysis of molecular variance established
the above ndings (Table 4). All components of molecular variance
were signicant at P = 0.001.
3.3. Combined RAPD and ISSR analysis
Two different types of markers were employed for assessment
of genetic diversity among and within different agro climate zones
of C. longa. A total of 160 bands were amplied with all the markers, out of which 126 were polymorphic, 34 were monomorphic
(Table 2). On the basis of combined data generated from RAPD, and
ISSR marker analysis, a dendrogram was constructed (Fig. 5). All
the 60 samples were separated into two distinct clusters of one
with only one sample and other with 59 samples each having a
common node at 61% similarity level. The 2nd cluster was further
separated into 2 subclusters having 13 samples in 1st subcluster
and 46 samples in 2nd subcluster. These subclusters shared a common node at 62.8% similarity level. The 2nd subcluster was further
divided into 2 groups, one with only 1 sample and other with 42
samples having common node at 63.5% similarity level. Total number of polymorphic bands was highest (PPB = 67.5%) in P9 region of
Orissa and lowest (42.50%) in P7 region. Similarly, Neis gene diversity (H = 0.257 0.1977) and Shannons Index (I = 0.3805 0.2811)
were also highest in P9 region. The lowest H and I values were

Fig. 5. Dendrogram showing the clustering pattern among 60 samples of turmeric

as revealed through Combined markers.

Neis genetic
diversity (H)
(mean SD)
0.2000
0.2219
0.2464
0.2469
0.2557
0.2438
0.1812
0.1992
0.2571
0.2305

0.2153
0.2142
0.2119
0.1982
0.2050
0.2035
0.2153
0.2169
0.1977
0.2138

0.2833 0.1931

No. of
polymorphic
loci

Percentage of
polymorphic loci
(PPB)

0.3082
0.3059
0.3013
0.2816
0.2910
0.2909
0.3085
0.3108
0.2811
0.3346

77
87
97
106
104
100
68
75
108
88

48.12
54.38
60.62
66.25
65.00
62.50
42.50
55.00
67.50
46.88

0.4192 0.2679

123

76.88

Shannons
information index
(I) (mean SD)
0.2904
0.3233
0.3592
0.3667
0.3753
0.3591
0.2619
0.2881
0.3805
0.3072

recorded for P7 region (Table 6). The mean observed number of

alleles (Na) was 1.76; the maximum (1.675 0.469) and minimum
(0.501 0.148) being for P9 and P1 regions (Table 5). The total genotype diversity (Ht) value was maximum (0.257) in P9 and minimum
(0.181) in P7 regions (Table 6). The partitioning of variations within
and among populations was analyzed by AMOVA, which revealed
occurrence of 48% of total genetic variations among the different
regions. It implies that remaining 52% was due to variations within
a region (Table 4). Analysis using combined markers, i.e., RAPD
and ISSR revealed that P9 (Western central table land) exhibited
relatively higher genetic diversity compared to other 9 regions.
4. Discussion
4.1. Polymorphism and genetic relationships among different
taxa of turmeric
Detection of high polymorphism and resolving power of marker
system used in this study are of considerable signicance. Amplication of large number of polymorphic bands indicated that the
primer sets used in this study could be of signicance for the assessment of genetic diversity in turmeric cultivars. Our results reect
similar ndings as reported earlier for the study of the genetic
variation in other species at cultivar level by using RAPD markers (Huang et al., 2003). In present study, high genetic distances
(max 0.8) and highest no of effective alleles (all multipurpose population 1.7688 0.42) is observed which reveals relatively high
genetic variation among the accessions. The overall genetic diversity among all populations of C. longa was high possibly due to a
wide range of ecological conditions within the distribution area of
its populations. In our study, signicant polymorphism (all multipurpose population 76.8%) observed among the populations
illustrates the genetic divergence among the turmeric samples
even though the cultivars are of the same origin. The cause for
the high level of polymorphism could be intra-specic variation
as reported by (Nayak et al., 2006), who found similar outcome
from RAPD analysis of 17 cultivars of turmeric. Several studies on
assessment of genetic diversity of plants using molecular markers have established the correlation between geographical distance
and genetic similarity between individuals (Islam, 2004). In the
present study, dendrogram for all 60 samples using RAPD and ISSR
combined markers showed interesting pattern formation of 2 clusters (I, major including 59 samples and II, including only 1 sample).
Cluster II includes accession P4-4 from Kandarpur is completely
separated from rest of the samples might be due to change in
edaphic factor since the area is situated in Mahanadi delta region
having high content of silt in the soil which enhances the soil fertility. Major cluster (I) was further subdivided into 5 subclusters
with mixed clustering pattern, i.e. no signicant population wise

290

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

segregation. The accession P4-6 (Puri) and P5-7 (Parlekhumundi)

showed maximum similarity of 83% and accession P6-4 (Pottangi,
Koraput) and P8-4 (Kandhamal) showed similarity of 82% belonging to two different geographical regions. Dendrograms did not
show any signicant pattern of clustering according to the locations from where genotypes were collected, indicating little or no
location specicity among turmeric genotypes. Similar results were
reported in Azuki bean (Yee et al., 1999), in groundnut (Dwivedi
et al., 2001) and in castor (Gajera et al., 2010). Some probable reasons may be assigned for the above inferences that, either turmeric
being propagated through rhizome as seed material, or there must
have occurred a chance migration of rhizome seed material by
growers from one region to another neighboring region or growers from different agro climatic zones must be sharing a common
major market for procuring of high yielding planting materials for
turmeric farming.
4.2. Genetic diversity among accessions of different agro climatic
zones
The result of Neis genetic diversity (H) (Tables 3, 5 and 6) generated from the POPGEN analysis shows relatively low genetic
diversity in turmeric population of P7 (South eastern ghat), P8
(Western undulating zone) with 0.181 and 0.199 respectively
whereas highest genetic diversity with 0.257 has been observed
in P9 zone (Western central table land). The present result of
each population indicated that the low level of genetic diversity
in hilly areas of P7 and P8 zone is existing because these hilly
populations are reasonably undisturbed and possess wider ecological adaptations such as open and shady places of forest margins,
whereas the plain land and plateau land populations of P9 comprising ecologically rather homogeneous condition experienced with
high disturbance due to intensive agricultural practices. Highest
Shannons informative indices (0.38) representing genetic heterogeneity and highest Neis genetic diversity (0.25) revealed through
combined markers was obtained for turmeric accessions collected
from climatic zone P9 (Western central table land). This indicates
better adaptation of accessions to varied range of environmental factors. Thus P9 accessions may be recommended for use in
several breeding programs and also as suitable cultivars for largescale cultivation by potential growers. The variability reected in
the germplasm of turmeric could be due to adaptability of crop
germplasm in different agro-ecological climatic conditions (Singh
et al., 1998). However, turmeric accessions of agro climatic zone P7
(South eastern ghat) were found to have lowest Shannons informative indices (0.26) and lowest Neis genetic diversity (0.18) may
be advocated selectively for developing special strategies to conserve these accessions as these are less adapted to the various
environmental conditions. (Islam, 2004) have also recommended
conservation prioritization for C. zedoaria population of plain land
and plateau lands showing low genetic diversity among them.
In the present study estimation of genetic diversity of turmeric
genotypes from different agroclimatic regions has therefore been
recognized as elementary topic to outline in situ and ex situ conservation strategies. The ISSR method has been reported to be
more reproducible (Goulao and Oliveira, 2001) and produces more
complex marker patterns than the RAPD approach (Chowdhury
et al., 2002), which is advantageous for differentiation of closely
related cultivars. ISSR has been used for cultivar identication in
numerous plant species, including rice (Joshi et al., 2000), apple
(Goulao and Oliveira, 2001), and strawberry (Arnau et al., 2003).
Some researchers have considered RAPD markers to represent
segments of DNA with non coding regions and to be selectively
neutral (Landergott et al., 2001), and some studies have shown
that RAPD markers are distributed throughout the genome and
may be associated with functionally important loci (Penner, 1996).

However, there is a little information to indicate that ISSR markers are functionally important (Esselman et al., 1999). Our results
signify the presence of great genetic variability among elite genotypes of turmeric. Both RAPD and ISSR markers are useful in the
assessment of turmeric diversity, the detection of duplicate sample in genotype collection, and the selection of a core collection to
enhance the efciency of genotype management for use in turmeric
breeding and conservation.
Acknowledgements
The authors are grateful to Prof (Dr.) S.C. Si, Dean, Centre of
Biotechnology and Prof (Dr.) M.R. Nayak, President, Siksha O
Anusandhan University for providing nancial support and encouraging throughout.
References
Antunes, L.M.G., Araujo, M.C.P., 2000. Mutagenicidade e antimutagenicidade dos
principais corantes para alimentos. Rev. Nutr. 13, 8188.
Araujo, C.C., Leon, L.L., 2001. Biological activities of Curcuma longa L. Mem. Inst.
Oswaldo Cruz 96, 723728.
Arnau, G., Lallemand, J., Bourgoin, M., 2003. Fast and reliable strawberry cultivar
identication using inter simple sequence repeat (ISSR) amplication. Euphytica
129, 6979.
Ceclio-Filho, A.B., Souza, R.J., Braz, L.T., Tavares, M., 2000. Crcuma: planta medicinal, condimentar e de outros usos potenciais. Cienc. Rural 30, 171175.
Chalmers, K.Y., Newton, A.C., Waugh, R., Powell, W., 1994. Evaluation of the extent of
genetic variation in mahoganies (Meliaceae) using RAPD markers. Theor. Appl.
Genet. 89, 504508.
Chaudhary, A.S., Sacham, S.K., Singh, R.L., 2006. Studies on varietal performance of
turmeric (Curcuma longa L.). Indian J. Crop Sci. 1, 189190.
Cheng, Z., Huang, H., 2009. SSR ngerprinting Chinese peach cultivars and Iandraces (Prunus persica) and analysis of their genetic relationships. Sci. Hort. 120,
188193.
Chowdhury, M.A., Vandenberg, B., Warkentin, T., 2002. Cultivar identication and
genetic relationship among selected breeding lines and cultivars in chickpea
(Cicer arietinum L.). Euphytica 127, 317325.
Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure for small quantities of
fresh leaf tissue. Phytochem. Bull. 19, 1115.
Dwivedi, S.L., Gurtu, S., Chandra, S., Yuejin, W., Nigam, S.N., 2001. Assessment of
genetic diversity among selected groundnut germplasm. I: RAPD analysis. Plant
Breed 120 (4), 345349.
Ebrahimi, R., Zamani, Z., Kashi, A., 2009. Genetic diversity evaluation of wild Persian
shallot (Allium hirtifolium Boiss.) using morphological and RAPD markers. Sci.
Hort. 119, 345351.
Esselman, E.J., Li, J.Q., Crawford, D., Winduss, J.L., Wolfe, A.D., 1999. Clonal diversity in
the rare Calamagrostis porteri ssp. Insperata (Poaceae): comparative results for
allozymes and random amplied polymorphic DNA (RAPD) and inter-simple
sequence repeat (ISSR) markers. Mol. Ecol. 8, 443451.
Excofer, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred
from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479491.
Gajera, B.B., Kumar, N., Singh, A.S., Punvar, B.S., Ravikiran, R., Subhash, N., Jadeja,
G.C., 2010. Assessment of genetic diversity in castor (Ricinus communis L.) using
RAPD and ISSR markers. Ind. Crops Prod. 32, 491498.
Ghislain, M., Zhang, D., Fazardo, D., Huamann, Z., Hismans, R.H., 1999. Marker
assisted sampling of the cultivated andean potato Solanum fureja collection using
RAPD markers. Genetic Res. Crop Evol. 46, 547555.
Goulao, L., Oliveira, C.M., 2001. Molecular characterization of cultivars of apple
(Malus domestica Borkh.) using microsatellite (SSR and ISSR) markers. Euphytica
122, 8189.
Huang, Y., Zhang, C.Q., Li, D.Z., 2003. Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):
implications for its conservation. J. Syst. Evol. 47 (2), 103109.
Islam, A., 2004. Genetic diversity of the genus Curcuma in Bangladesh and further
biotechnological approaches for in vitro regeneration and long-term conservation of C. longa germplasm. PhD thesis, University of Hannover.
Joshi, S.P., Gupta, V.S., Aggarwal, R.K., Ranjekar, P.K., 2000. Genetic diversity and
phylogenetic relationship as revealed by inter simple sequence repeat (ISSR)
polymorphism in the genus Oryza. Theor. Appl. Genet. 100, 13111320.
Landergott, U., Holderegger, R., Kozlowski, G., Schneller, J.J., 2001. Historical bottlenecks decrease genetic diversity in natural populations of Dryopteris cristata.
Heredity 87, 344355.
Miller, M.P., 1998. AMOVA-PREP 1.01: A Program for the Preparation of the AMOVA
Input Files from Dominant-Marker Raw Data. Department of Biological Sciences,
Northern Arizona University, Flagstaff, AZ.
Nass, L.L., 2001. Utilizaco de recursos genticos vegetais no melhoramento. In: Nass,
L.L., Valois, A.C.C., Melo, I.S., Valadares-Inglis, M.C. (Eds.), Recursos Genticos e
MelhoramentoPlantas. Fundaco MT, Rondonpolis, pp. 2956.

S. Singh et al. / Industrial Crops and Products 37 (2012) 284291

Nayak, S., Naik, P.K., Acharya, L.K., Pattnaik, A.K., 2006. Detection and evaluation of
genetic variation in 17 promising cultivars of turmeric (Curcuma longa L.) using
4C nuclear DNA content and RAPD markers. Cytologia (Tokyo) 71 (1), 4955.
Nei, M., 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad.
Sci. U. S. A. 70, 33213323.
Penner, G.A., 1996. RAPD analysis of plant genomes. In: Jauhar, P.P. (Ed.), Methods
of genome analysis in plants. CRC press, Boca Raton, pp. 251268.
Prevost, A., Wilkins, M.J., 1999. A new system for comparing PCR primers applied to
ISSR ngerprinting of potato cultivars. Theor. Appl. Genet. 98, 661668.
Ravindran, P.N., 2007. Turmericthe golden spice of life. In: Ravindran, P.N., Babu,
K.N., Sivarman, K. (Eds.), Turmeric the Genus Curcuma. CRC Press, Boca Raton,
pp. 115.
Sasikumar, B., 2005. Genetic resources of Curcuma: diversity, characterization and
utilization. Plant Genet. Res. 3, 230251.
Singh, A.K., Smartt, J., Simpson, C.E., Raina, S.N., 1998. Genetic variation vis--vis
molecular polymorphism in groundnut, Arachis hypogaea L. Genet. Resour. Crop
Evol. 45, 119126.

291

Smith, J.S.C., Chin, E.C.L., Shu, H., Smith, O.S., Wall, S.J., Senior, M.L., Mitchell, S.E.,
Kresovich, S., Ziegle, J., 1997. An evaluation of the utility of SSR loci as molecular
markers in maize (Zea mays L.): comparison of data fro RFLPs and pedigree.
Theor. Appl. Genet. 95, 163173.
Sneath, P.H.A., Sokal, R.R., 1973. Numerical Taxonomy. Freeman, San Francisco, CA,
p. 573.
Thimmappaiah, W.G., Santhosh, D.S., Melwyn, G.S., 2009. Assessment of genetic
diversity in cashew germplasm using RAPD and ISSR markers. Sci. Hort. 120,
411417.
Williams, J.G.K., Kubelik, A.R., Liva, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphisms amplied by arbitrary primers are useful as genetic markers. Nucleic
Acids Res. 18, 65316535.
Yee, E., Kidwell, K.K., Sills, G.R., Lumpkin, T.A., 1999. Diversity among selected Vigna
angularis (Azuki) accessions on the basis of RAPD and AFLP markers. Crop Sci.
39, 268275.
Zeitkiewicz, E., Rafalski, A., Labuda, D., 1994. Genome nger printing by simple
sequence repeat (SSR)-anchored PCR amplication. Genomics 20, 176183.