Saudi Journal of Biological Sciences (2015 , 159–16

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.co

ORIGINAL ARTICLE

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 Abstract
Zingiber officinale; The
RAPD; present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from
UPGMA; subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers
India screened were informative and produced 275 amplification products, among which 261 products (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
Ginger (Zingiber officinale Roscoe) Fam. Zingiberaceae is a valued the world’s leading producers (Blumenthal et al., 2000). India
medicinal crop and has been used as a spice for over recorded 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

* Corresponding author. Mobile: +91 9212050090. its broad spectrum of biological and pharmacological applications
E-mail address: drmmujeeb@rediffmail.com (M. Mujeeb). including antitumor, antioxidant, anti-inflamma-
Peer review under responsibility of King Saud University.
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).
 160
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 genotype
identification (Wilde et al., 1992; Koller et al., 1993; Wolff and
Peters-Van Run, 1993).
K. Ashraf et al.
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 Na 2 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 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
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India 161
buffer (Promega; 20 mM Tris–HCl (pH 8.4); 50 mM KCl), 1.5 to form a binary data matrix. The commercial software package
mM MgCl2, 300 lM each of deoxynucleotide triphosphate NTSYS-PC version 2.0 (Rohlf, 1995) was used to develop a
(dNTP), 25 pM decanucleotide primer (Operon similarity matrix. These data were used to construct dendogram
TechnologyInc.,USA),1unitTaqDNApolymerase(Promega) for cluster analysis based on unweighted pair group method with
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
Table 2 Trivandrum (Kerala)
Optical density 829015
of DNA isolated from 00
N, 7657accessions
different 9 E
0 00
of Local farmer,
Zingiber CultivatedRosc.
officinale 7th Nov. 2011
G8
Code No. Delhi (Delhi) Zingiber officinale2836 0
3600N, 771304800E Optical
samples Herbal garden,
density ( k) Hamdard University, Cultivated 19th
RatioDec. 2011 nm
of 260/280
G9 Chandigarh (Haryana) 3045 0 N, 764604800E
0 00
Local farmer, Cultivated 17th Oct. 2011
260 nm 280 nm
G10 Bhopal (M.P.) 23150000N, 77250000E Local farmer, Cultivated 18th Jan. 2012
G1
G11 Guwahati (Assam)Patna (Bihar) 0.094Local farmer, Cultivated
0.051 1.84 22nd Nov. 2011
26110000N, 91440000E
G2 Lucknow (U.P.) 0.034 0.019 1.78
G12 Surat (Gujrat) 26110000N, 91440000E Keshal nursery, Cultivated 5th Jan. 2012
G3 Dehradun (Utrakhand) 0.032 0.017 1.88
G4 Erode (Tamil Nadu) 0.093 0.052 1.78
G5 Bangalore (Karnataka) 0.087 0.045 1.92
G6 Nashik (Maharashtra) 0.034 0.016 1.80
G7 Trivandrum (Kerala) 0.051 0.016 1.88
G8 Delhi (Delhi) 0.040 0.021 1.90
G9 Chandigarh (Haryana) 0.070 0.037 1.89
G10 Bhopal (M.P.) 0.088 0.047 1.87
G11 Guwahati (Assam) 0.092 0.051 1.80
G12 Surat (Gujrat) 0.020 0.011 1.81

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 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 N AB/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 resulting similarity
coefficients were employed to assess the relationship among
ginger accessions. All amplified profiles were analyzed together
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 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 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
162
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
K. Ashraf et al.
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

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.

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
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India 163

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.

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.
 164 K. Ashraf et al.
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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