2 , 159–16

Genetic diversity analysis of Zingiber Officinale

Roscoe by RAPD collected from subcontinent of India
Kamran Ashraf a, Altaf Ahmad b, Anis Chaudhary b, Mohd. Mujeeb a,*, Sayeed
Ahmad a, Mohd. Amir a, N. Mallick a
a
Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New
Delhi 10062, India
b Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 10062, India

Received 18 April 2013; revised 7 September 2013; accepted 10 September 2013 Available
online 17 September 2013

KEYWORDS
Zingiber officinale; Abstract The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc.
RAPD; collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of
UPGMA; twenty primers screened were informative and produced 275 amplification products, among which 261 products
India (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23%
to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275
RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of
UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions.
This investigation was an understanding of genetic variation within the accessions. It will also provide an important
input into determining resourceful management strategies and help to breeders for ginger improvement program. ª
2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introductions 2000 years (Bartley and Jacobs, 2000). It is cultivated in many

tropical and subtropical countries in which, China and India are the
Ginger (Zingiber officinale Roscoe) Fam. Zingiberaceae is a valuedworld’s leading producers (Blumenthal et al., 2000). India recorded
medicinal crop and has been used as a spice for over the highest production of ginger in the world
(0.38 million tons) in 2009 (Sajeeva et al., 2011).The impor-

tance of ginger is gaining recently because of its low toxicity and

its broad spectrum of biological and pharmacological applications
* Corresponding author. Mobile: +91 9212050090.
including antitumor, antioxidant, anti-inflamma-
E-mail address: drmmujeeb@rediffmail.com (M. Mujeeb).
Peer review under responsibility of King Saud University.
 160
tory, antiapoptotic, cytotoxic, anti-proliferative and anti-platelet
activities (Sekiwa et al., 2000; Shukla and Singh, 2007; Wei et al.,
2005; Young et al., 2005).
1319-562X ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.09.005
The ginger rhizome contains various biologically active
compounds such as gingerol, shogaol, ginger protease, capsaicin
and several sesquiterpenes like zingiberol, zingiberenol and these
constituents may vary depending on the place of origin and
whether the rhizomes are fresh or dry (Tang and Eisenbrand,
1992; Ali et al., 2008). Over 50 components of the oil present in
ginger are mainly monoterpenoids [b-phellandrene, (+)-
camphene, cineole, geraniol etc.] and sesquiterpenoids [a
zingiberene (30–70%), b-sesquiphellandrene (15–20%),
bbisabolene (10–15%), (E-E)-a-farnesene, arcurcumene, and
zingiberol] (Langner et al., 1998; Evans, 2002).The phenolic
ketone compounds such as 6-gingerol, 8-gingerol and 10-gingerol
are the principle active pungent compounds (Connell and
Sutherland, 1969). The pungency of fresh ginger is primarily due
to the gingerols, which are a homologous series of phenols. The
pungency of dry ginger mainly results from shogaols which are
the dehydrated forms of gingerols. Shogaols are formed from the
corresponding gingerol during thermal processing (Wohlmuth et
al., 2005). Ginger protease plays a very important role in meat
tenderization (10-fold activity) and it can significantly improve
the flavor as well as quality of the meat by increasing nutritious
value (Naveena and Mendiratta, 2001; Zhou et al., 1996). Ginger
protease can also improve the quality of food processing without
reducing the nutritious value. It also separates casien protein into
smaller peptide (Song et al., 2001). It is reported that variations in
quantity and quality of the polyphenols present in plant foods
occurred due to different factors, such as plant genetics and
cultivar, soil composition and growing conditions, maturity state,
and post harvest conditions (Jaffery et al., 2003). Besides this,
most of the crop improvement programs of ginger are restricted to
the assessment and selection of naturally occurring clonal
variations (Rout et al., 1998; Palai and Rout, 2007).Therefore,
diversity analysis and identification of genetically distant clones
or genotypes are vital to the ginger improvement program.
The assessment of genetic diversity may be done within and
between populations at molecular level by using various
techniques like allozymes or DNA analysis (Mondini et al., 2009).
During the past decades, use of molecular markers is gaining
attention to reveal polymorphisms at the DNA level. Different
marker based techniques are available for the identification of
plants. Out of these, molecular marker based is more accepted
because it overcomes many of the limitations of morphological
and biochemical techniques since they are not affected by the
environmental or developmental stage and can detect a variation
at the DNA level (Tingey and Tufo, 1993). PCR-based molecular
markers were extensively used in many plant species for
identification, phylogenetic analysis, population studies and
genetic linkage mapping. RAPD markers are markers of choice,
because of its simplicity and low-cost nature, rapid, inexpensive
and effective system for studying plant genetic relationships
(Williams et al., 1990). The RAPD markers could also be used in
the study of genetic variability of species or natural populations
(Lashermes et al., 1993; Wilkie et al., 1993) and in the study of
K. Ashraf et al.
genotype identification (Wilde et al., 1992; Koller et al., 1993;
Wolff and Peters-Van Run, 1993).
Till date several studies have been reported on genetic
diversity study of different ginger varieties in India but to our
knowledge very less or no study has ever been reported on
diversity collected from all major ecological zones or provinces
of India. Since ginger is a very poorly studied crop and its
molecular information is limited, hence it is imperative to know
the genetic diversity among different accessions from Indian
subcontinent.
2. Materials and methods
2.1. Plant material
The crude drug samples of rhizomes of Z. officinale were collected
in the months of October (2011) – January (2012) from twelve
different regions (Table 1) of India. All collected samples were
authenticated by the taxonomist Professor M.P. Sharma,
Department of Botany, Faculty of Science, Jamia Hamdard, New
Delhi. All voucher specimens were deposited in the Molecular
Ecology Laboratory, Jamia Hamdard, New Delhi, India.
2.2. Genetic diversity analysis
2.2.1. Isolation of genomic DNA
Conventional DNA isolation protocol suggests the use of leaves
which are devoid of secondary metabolite and this metabolite causes
hindrance in DNA isolation and makes the whole process tedious.
So we developed CTAB modified method. In brief, genomic DNA
of frozen rhizome samples was isolated by modified cetyl trimethyl
ammonium bromide (CTAB) extraction method (Doyle and Doyle,
1990). 1 g of rhizome sample along with polyvinylpyrrolidone was
triturated in liquid nitrogen to fine powder. 5 ml of 1% CTAB (100
mM Tris–HCl buffer pH 8.0, 1.4 M NaCl, 20 mM, EDTA, 1%
mercaptoethanol) buffer was added to the homogenate, and
centrifuged at 10,000 rpm for about 15 min. Added, 2 vol. of 2%
CTAB (100 mM Tris–HCl buffer, 1.4 M NaCl, 20 mM
EDTA) to the collected aqueous phase. This mixture was incubated
at 65 C for about 60 min with intermittent shaking. The suspension
was then cooled to room temperature and equal volume of
chloroform and isoamyl alcohol (24:1) was added. The mixture was
then centrifuged at 10,000 rpm for 15 min. The aqueous phase was
collected, and to it was added 0.6 volume of cold isopropanol and
1/30 volume of sodium acetate (3 M, pH 5.2) and incubated at 20 C
for 1 h. The sample was centrifuged at 10,000 rpm for 15 min to
obtain the DNA pellet. The pellet was washed with 80% ethanol
twice, air dried and dissolved in TE buffer (10 mM Tris buffer, pH
8.0, 1 mM Na2 EDTA). The isolated DNA was treated with RNase
A (10 lg/ml) at 37 C for 30 min. DNA concentration and purity were
determined by measuring the absorbance of diluted DNA solution at
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India
260 nm and 280 nm. The quality of the DNA was determined using
agarose gel electrophoresis stained with ethidium bromide.
2.2.2. RAPD amplification
Polymerase chain reaction (PCR) was performed according to the
method based on Williams et al. (1990). PCR reaction was carried
out in 25 ll reaction tubes. Amplification reaction contained PCR
buffer (Promega; 20 mM Tris–HCl (pH 8.4); 50 mM KCl), 1.5 mM
MgCl2, 300 lM each of deoxynucleotide triphosphate (dNTP), 25
pM decanucleotide primer (Operon
TechnologyInc.,USA),1unitTaqDNApolymerase(Promega)
161
other and data were scored as the absence (0) or presence (1) of a
DNA band for each of the primer-accession combination. The size
of amplified DNA fragments was estimated by comparison with the
molecular weight marker 100bp–1500pb DNA Ladder (Bio-Basic.
Inc.). Pair-wise comparisons of all ginger accessions based on
absence or presence of unique and shared DNA bands were used to
make similarity coefficients. Estimates of genetic similarity (S) were
calculated between all pairs of ginger accessions using the formula,
S = 2 NAB/NA + NB (Nei and Li, 1979). Where, NAB is the number of
amplified products common to both A and B. NA and NB correspond
to the number of amplified products in A and B respectively. The

Table 1 List of different accessions of Zingiber officinale used in the present study.

Code No. Cultivation regions (Provinces) Latitude, Altitude Source Date of collections
G1 Patna (Bihar) 2536039.600N, 858038.400E Local farmer, cultivated 23rd Oct. 2011
G2 Lucknow (U.P.) 2650049.200N, 8056049.200E Herbal garden, Integral University, Cultivated 25th Oct. 2011
G3 Dehradun (Utrakhand) 3018056.5200N, 78200.9600E Local farmer, Cultivated 28th Oct. 2011
G4 Erode (Tamil Nadu) 11210000N, 77440000E Local farmer, Cultivated 2nd Nov. 2011
G5 Bangalore (Karnataka) 12580000N, 77340000E Local farmer, Cultivated 4th Nov. 2011
G6 Nashik (Maharashtra) 2000000N, 734604800E Local farmer. Cultivated 12th Nov. 2011
G7 Trivandrum (Kerala) 82901500N, 76570900E Local farmer, Cultivated 7th Nov. 2011
G8 Delhi (Delhi) 283603600N, 771304800E Herbal garden, Hamdard University, Cultivated 19th Dec. 2011
G9 Chandigarh (Haryana) 30450000N, 764604800E Local farmer, Cultivated 17th Oct. 2011
G10 Bhopal (M.P.) 23150000N, 77250000E Local farmer, Cultivated 18th Jan. 2012
G11 Guwahati (Assam) 26110000N, 91440000E Local farmer, Cultivated 22nd Nov. 2011
G12 Surat (Gujrat) 26110000N, 91440000E Keshal nursery, Cultivated 5th Jan. 2012

Zingiber officinale k

0.094 0.051
0.034 0.019
0.032 0.017
0.093 0.052
0.087 0.045
0.034 0.016
0.051 0.016
0.040 0.021
0.070 0.037
0.088 0.047
0.092 0.051
0.020 0.011

and 50 ng of template genomic DNA. Amplification was performed
in a thermal cycler (Master Cycler, Eppendorf, USA) using the
following conditions: 40 cycles at 94 C for 3 min; 94 C for 30 s, 50
C for 1 min and 72 C for 2 min; final extension at 72 C for 5 min.
Amplification products were separated alongside a molecular
weight marker (100 bp plus ladder,
M/SBangaloreGenei)by1.2%agarosegelelectrophoresisin1·
TAE(TrisacetateEDTA)bufferstainedwithethidiumbromide and
visualized under UV light. Gel photographs were scanned through a
Gel Doc System (UVitech, USA) and the amplification product sizes
were evaluated using the Software Quantity One (Bio Rad, USA).
2.2.3. Data analysis
Data analysis was done using Alpha Imager EC software. For each
accession the amplified fragment/band was treated as a unit
character. Amplified agarose gel pictures were compared with each
resulting similarity coefficients were employed to assess the
relationship among ginger accessions. All amplified profiles were
analyzed together to form a binary data matrix. The commercial
software package NTSYS-PC version 2.0 (Rohlf, 1995) was used to
develop a similarity matrix. These data were used to construct
dendogram for cluster analysis based on unweighted pair group
method with arithmatic means (UPGMA) using Winbbot (IRRI, Los
Ban˜ os, Philippines) (Yap and Nelson, 1996).
3. Results and discussion
3.1. Genetic diversity analysis
Genetic variability studies in ginger collected from different
geographical regions of India have been carried out using RAPD
 162
markers. DNA was isolated by CTAB method. DNA extraction
of ginger proved difficult due to the presence of secondary
metabolites. A modified CTAB method by Doyle and Doyle
(1990) proved to be fruitful. The modified method included
higher incubation temperature (65 C). Random amplified
polymorphic DNA and related techniques require less DNA, but
purity is necessary to ensure repeatability and confidence (Welsh
and McClelland, 1990; Williams et al., 1990). The purity of DNA
determined from the ratio of optical density of 260/280 ratio
which ranged from 1.78 to 1.92 for the samples indicates the
purity of DNA in all samples (Table 2).
The present study offers an optimization of primer screening
for evaluation of genetic relationship among the twelve accessions
of ginger through RAPD analysis. Twenty decamer primers from
Operon Technologies (Alameda, California, USA) were initially
screened using one species of Delhi to determine the suitability of
each primer for the study. Primers were selected for further
analysis based on their ability to detect distinct and polymorphic
Figure 1 Dendrogram based on UPGMA (unweighted pair-group method with arithmetic averages) analysis of genetic similarities estimated among
the 12 accessions of Z. officinale by the means of 13 RAPD primers.
K. Ashraf et al.
amplified products within the species. Out of twenty decamer
random primers used for 12 accessions, 07 primers did not
produce any amplification at all in the initial screening while 13
primers showed amplified polymorphic pattern. These primers
were then used for RAPD analysis for 12 accessions. The
commercial software package NTSYS-PC (Rohlf, 1995) was used
to develop similarity matrix These data were then used for
constructing dendrogram (Fig. 1) for cluster analysis based on
Unweighed pair group method with arithmatic mean (UPGMA).
The selected primers generated distinctive products in the range
of 100–1500 bp. The maximum and minimum number of bands
was produced by the primers OPA-11(28), and OPA-06(12),
respectively (Table 3). A total number of 275 amplified fragments
was scored across twelve accessions of ginger for the selected
primers, and was used to estimate genetic relationships among
themselves. Out of 275 fragments obtained, 261 fragments
(94.90%) were
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India 163

Table 3 Primer sequence and amplified products of 12 accessions of Zingiber officinale Rosc.
Primer Sequence of primer (50 -30) Size of product amplified No. of amplified band No. Polymorphic band % Polymorphism
(bps) 100–1500 bps
OPAA -01 AGACGGCTCC 215–1181 21 20 95.23
OPA A-02 GAGACCAGAC 258–1153 21 19 90.47
OPA A-03 TTAGCGCCCC 135–844 14 13 92.87
OPAA -04 AGGACTGCTC 163–1300 25 24 96
OPAA -05 GGCTTTAGCC 253–1375 26 26 100
OPAA -06 TCAAGCTAAC 314–766 12 11 91.66
OPAA -07 CTACGCTCAC 250–1411 26 24 92.30
OPAA -08 TCCGCAGTAG 145–1125 26 26 100
OPAA -09 AGATGGGCAG 287–1400 21 21 100
OPAA -10 TGGTCGGGTG 428–1232 19 17 89.47
OPAA -11 ACCCGACCTG 292–1269 28 27 96.42
OPAA-12 GGACCTCTTG 280–988 17 15 88.23
OPAA-15 ACGGAAGCCC 354–1054 19 18 94.73
Figure 2 RAPD amplification pattern of different accessions of Z. officinale using primer OPA-10, M-molecular marker (100–1500 bps), G1-
Patna, G2-Lucknow, G3-Dehradun, G4-Erode, G5-Bangalore, G6-Nashik, G7-Trivandrum, G8-Delhi, G9-Chandigarh, G10-Bhopal, G11-
Guwahati and G12-Surat.
 164 K. Ashraf et al.

Figure 3 RAPD amplification pattern of different accessions of Z. officinale using primer OPA-12, M-molecular marker (100–1500 bps), G1-
Patna, G2-Lucknow, G3-Dehradun, G4-Erode, G5-Bangalore, G6-Nashik, G7-Trivandrum, G8-Delhi, G9-Chandigarh, G10-Bhopal, G11-
Guwahati and G12-Surat.
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India 165
Table 4 Similarity matrix table of 12 samples of Z. officinale.
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12

G1 1

G2 0.29 1

G3 0.28 0.28 1

G4 0.25 0.27 0.21 1

G5 0.22 0.23 0.25 0.29 1

G6 0.22 0.21 0.23 0.26 0.25 1

G7 0.21 0.22 0.24 0.32 0.27 0.27 1

G8 0.23 0.21 0.22 0.15 0.26 0.23 0.25 1

G9 0.23 0.19 0.22 0.22 0.24 0.22 0.24 0.39 1

G10 0.26 0.23 0.23 0.2 0.19 0.21 0.22 0.3 0.26 1

G11 0.17 0.21 0.2 0.15 0.19 0.19 0.21 0.22 0.19 0.23 1

G12 0.17 0.2 0.18 0.2 0.19
G1-Patna, G2-Lucknow, G3-Dehradun, G4-Erode, G5-Bangalore, G6-Nashik, G7-Trivandrum, G8-Delhi, G9-Chandigarh, G10-Bhopal, G11Guwahati
and G12-Surat.
polymorphic. The pattern of RAPD produced by the primers
OPA-10 and OPA-12 are shown in (Figs. 2 and 3).
Pair-wise genetic similarities ranged from 0.21 to 0.39 in all
accessions with a mean value of 0.30 (Table 4). The matrix values
indicated that these accessions were distantly related to each other
as reported by (Palai and Rout, 2007). Based on the dendrogram,
the 12 accessions were grouped into two main clusters (cluster I
and cluster II). Cluster I is divided into sub cluster IA and IB.
These subcluster contain Patna and Lucknow with similarity of
29%. Dehradun is similar with Patna and Lucknow with 28%. In
cluster IB, Erode and Trivandrum shows similarity of 32%. In
lower cluster II, maximum similarity is shown by Delhi and
Chandigarh with 39%. The similarity between Bhopal and Surat
is found to be 31%. Three pairs of ginger varieties show least
similarity among all accessions of Erode and Guwahati, Erode and
Delhi, and Nashik and Surat at 15%. The high difference in gene
diversity among accessions/varieties reveals the presence of
strong genetic structure between them and thus significant
differences exist in the genotypic diversity among themselves.
Our finding is supported by results of Mohd et al. (2004) who
reported that the genetic variation occurred among the three
ginger cultivars from Malaysia using a RAPD marker. RAPD
analysis has been found to be useful in differentiating closely
related species (Zhang et al., 2001).The genetic relation through
RAPD markers provides a reliable method for the identification
of varieties than morphological characters (Palai et al., 2007).
Islam et al. (2007) findings also supported our results that there a
high level of genetic diversity exists within Curcuma zedoaria
populations. There is a common trend of maintaining high genetic
diversity within populations in tropical plants as reported by
Hamrick and Loveless (1989). Our results are also confirmatory
with the finding of Huang et al. (2003) who reported significant
(high) genetic variations by RAPD markers in other species at
cultivar level. Our finding is also supported by Jatoi et al. (2008)
0.15 0.23 0.26 0.24 0.31 0.23 1
that there occurs a high degree of genetic variation in ginger
collected from the Asian regions.
4. Conclusions
The main emphasis of the present study was to assess the genetic
diversity at intra specific level among the 12 accessions of ginger
of Indian subcontinent using RAPD markers. RAPD analysis
shows that there is a high level of polymorphism among different
accessions. From this study, it was understood that each location
varied with respect to environmental factors and genetic
parameters. Results showed that the accessions whose cultivation
regions are very close shows maximum similarity among them as
compared to accessions which are farther apart. This outcome is
supported by Nayak et al. (2006) who established that main cause
of polymorphism could be intraspecific variation among different
cultivars. These findings will also provide an important
contribution in determining resourceful management strategies
for breeders for ginger improvement program.
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