Saudi Journal of Biological Sciences (2014) 21, 159165

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Genetic diversity analysis of Zingiber Ocinale

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 The present investigation was undertaken for the assessment of 12 accessions of Zingiber
Zingiber ofcinale; ofcinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using
RAPD; CTAB method. Thirteen out of twenty primers screened were informative and produced 275 ampli-
UPGMA; cation products, among which 261 products (94.90%) were found to be polymorphic. The percent-
India age 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 Jaccards similarity coefcients 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 pro-
vide an important input into determining resourceful management strategies and help to breeders
for ginger improvement program.
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

Ginger (Zingiber ofcinale Roscoe) Fam. Zingiberaceae is a
valued medicinal crop and has been used as a spice for over
* Corresponding author. Mobile: +91 9212050090.
E-mail address: drmmujeeb@rediffmail.com (M. Mujeeb).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
tropical and subtropical countries in which, China and India
are the worlds leading producers (Blumenthal et al., 2000). In-
dia 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 its broad spectrum of biological and pharmacological
applications including antitumor, antioxidant, anti-inamma-
tory, antiapoptotic, cytotoxic, anti-proliferative and anti-plate-
let activities (Sekiwa et al., 2000; Shukla and Singh, 2007; Wei
et al., 2005; Young et al., 2005).

1319-562X 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
160 K. Ashraf et al.

The ginger rhizome contains various biologically active inces of India. Since ginger is a very poorly studied crop and its
compounds such as gingerol, shogaol, ginger protease, capsai- molecular information is limited, hence it is imperative to
cin and several sesquiterpenes like zingiberol, zingiberenol and know the genetic diversity among different accessions from In-
these constituents may vary depending on the place of origin dian subcontinent.
and whether the rhizomes are fresh or dry (Tang and Eisen-
brand, 1992; Ali et al., 2008). Over 50 components of the oil
present in ginger are mainly monoterpenoids [b-phellandrene, 2. Materials and methods
(+)-camphene, cineole, geraniol etc.] and sesquiterpenoids [a
zingiberene (3070%), b-sesquiphellandrene (1520%), b- 2.1. Plant material
bisabolene (1015%), (E-E)-a-farnesene, arcurcumene, and
zingiberol] (Langner et al., 1998; Evans, 2002).The phenolic The crude drug samples of rhizomes of Z. ofcinale were col-
ketone compounds such as 6-gingerol, 8-gingerol and 10-ging- lected in the months of October (2011) January (2012) from
erol are the principle active pungent compounds (Connell and twelve different regions (Table 1) of India. All collected sam-
Sutherland, 1969). The pungency of fresh ginger is primarily ples were authenticated by the taxonomist Professor M.P.
due to the gingerols, which are a homologous series of phenols. Sharma, Department of Botany, Faculty of Science, Jamia
The pungency of dry ginger mainly results from shogaols Hamdard, New Delhi. All voucher specimens were deposited
which are the dehydrated forms of gingerols. Shogaols are in the Molecular Ecology Laboratory, Jamia Hamdard, New
formed from the corresponding gingerol during thermal pro- Delhi, India.
cessing (Wohlmuth et al., 2005). Ginger protease plays a very
important role in meat tenderization (10-fold activity) and it 2.2. Genetic diversity analysis
can signicantly improve the avor as well as quality of the
meat by increasing nutritious value (Naveena and Mendiratta, 2.2.1. Isolation of genomic DNA
2001; Zhou et al., 1996). Ginger protease can also improve the
Conventional DNA isolation protocol suggests the use of
quality of food processing without reducing the nutritious va-
leaves which are devoid of secondary metabolite and this
lue. It also separates casien protein into smaller peptide (Song
metabolite causes hindrance in DNA isolation and makes the
et al., 2001). It is reported that variations in quantity and qual-
whole process tedious. So we developed CTAB modied meth-
ity of the polyphenols present in plant foods occurred due to
od. In brief, genomic DNA of frozen rhizome samples was iso-
different factors, such as plant genetics and cultivar, soil com-
lated by modied cetyl trimethyl ammonium bromide (CTAB)
position and growing conditions, maturity state, and post har-
extraction method (Doyle and Doyle, 1990). 1 g of rhizome
vest conditions (Jaffery et al., 2003). Besides this, most of the
sample along with polyvinylpyrrolidone was triturated in li-
crop improvement programs of ginger are restricted to the
quid nitrogen to ne powder. 5 ml of 1% CTAB (100 mM
assessment and selection of naturally occurring clonal varia-
TrisHCl buffer pH 8.0, 1.4 M NaCl, 20 mM, EDTA, 1%
tions (Rout et al., 1998; Palai and Rout, 2007).Therefore,
mercaptoethanol) buffer was added to the homogenate, and
diversity analysis and identication of genetically distant
centrifuged at 10,000 rpm for about 15 min. Added, 2 vol. of
clones or genotypes are vital to the ginger improvement
2% CTAB (100 mM TrisHCl buffer, 1.4 M NaCl, 20 mM
program.
EDTA) to the collected aqueous phase. This mixture was incu-
The assessment of genetic diversity may be done within
bated at 65 C for about 60 min with intermittent shaking. The
and between populations at molecular level by using various
suspension was then cooled to room temperature and equal
techniques like allozymes or DNA analysis (Mondini et al.,
volume of chloroform and isoamyl alcohol (24:1) was added.
2009). During the past decades, use of molecular markers is
The mixture was then centrifuged at 10,000 rpm for 15 min.
gaining attention to reveal polymorphisms at the DNA level.
The aqueous phase was collected, and to it was added 0.6 vol-
Different marker based techniques are available for the iden-
ume of cold isopropanol and 1/30 volume of sodium acetate
tication of plants. Out of these, molecular marker based is
(3 M, pH 5.2) and incubated at 20 C for 1 h. The sample
more accepted because it overcomes many of the limitations
was centrifuged at 10,000 rpm for 15 min to obtain the DNA
of morphological and biochemical techniques since they are
pellet. The pellet was washed with 80% ethanol twice, air dried
not affected by the environmental or developmental stage
and dissolved in TE buffer (10 mM Tris buffer, pH 8.0, 1 mM
and can detect a variation at the DNA level (Tingey and
Na2 EDTA). The isolated DNA was treated with RNase A
Tufo, 1993). PCR-based molecular markers were extensively
(10 lg/ml) at 37 C for 30 min. DNA concentration and purity
used in many plant species for identication, phylogenetic
were determined by measuring the absorbance of diluted DNA
analysis, population studies and genetic linkage mapping.
solution at 260 nm and 280 nm. The quality of the DNA was
RAPD markers are markers of choice, because of its simplic-
determined using agarose gel electrophoresis stained with ethi-
ity and low-cost nature, rapid, inexpensive and effective sys-
dium bromide.
tem for studying plant genetic relationships (Williams et al.,
1990). The RAPD markers could also be used in the study
2.2.2. RAPD amplication
of genetic variability of species or natural populations
(Lashermes et al., 1993; Wilkie et al., 1993) and in the study Polymerase chain reaction (PCR) was performed according to
of genotype identication (Wilde et al., 1992; Koller et al., the method based on Williams et al. (1990). PCR reaction was
1993; Wolff and Peters-Van Run, 1993). carried out in 25 ll reaction tubes. Amplication reaction con-
Till date several studies have been reported on genetic tained PCR buffer (Promega; 20 mM TrisHCl (pH 8.4);
diversity study of different ginger varieties in India but to 50 mM KCl), 1.5 mM MgCl2, 300 lM each of deoxynucleotide
our knowledge very less or no study has ever been reported triphosphate (dNTP), 25 pM decanucleotide primer (Operon
on diversity collected from all major ecological zones or prov- Technology Inc., USA), 1 unit Taq DNA polymerase (Promega)
Genetic diversity analysis of Zingiber Ofcinale Roscoe by RAPD collected from subcontinent of India 161

Table 1 List of different accessions of Zingiber ofcinale used in the present study.
Code No. Cultivation regions (Provinces) Latitude, Altitude Source Date of collections
G1 Patna (Bihar) 25360 39.600 N, 8580 38.400 E Local farmer, cultivated 23rd Oct. 2011
G2 Lucknow (U.P.) 26500 49.200 N, 80560 49.200 E Herbal garden, Integral University, Cultivated 25th Oct. 2011
G3 Dehradun (Utrakhand) 30180 56.5200 N, 7820 0.9600 E Local farmer, Cultivated 28th Oct. 2011
G4 Erode (Tamil Nadu) 11210 000 N, 77440 000 E Local farmer, Cultivated 2nd Nov. 2011
G5 Bangalore (Karnataka) 12580 000 N, 77340 000 E Local farmer, Cultivated 4th Nov. 2011
G6 Nashik (Maharashtra) 2000 000 N, 73460 4800 E Local farmer. Cultivated 12th Nov. 2011
G7 Trivandrum (Kerala) 8290 1500 N, 76570 900 E Local farmer, Cultivated 7th Nov. 2011
G8 Delhi (Delhi) 28360 3600 N, 77130 4800 E Herbal garden, Hamdard University, Cultivated 19th Dec. 2011
G9 Chandigarh (Haryana) 30450 000 N, 76460 4800 E Local farmer, Cultivated 17th Oct. 2011
G10 Bhopal (M.P.) 23150 000 N, 77250 000 E Local farmer, Cultivated 18th Jan. 2012
G11 Guwahati (Assam) 26110 000 N, 91440 000 E Local farmer, Cultivated 22nd Nov. 2011
G12 Surat (Gujrat) 26110 000 N, 91440 000 E Keshal nursery, Cultivated 5th Jan. 2012

and 50 ng of template genomic DNA. Amplication 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; nal
extension at 72 C for 5 min. Amplication products were sepa-
rated alongside a molecular weight marker (100 bp plus ladder,
M/S Bangalore Genei) by 1.2% agarose gel electrophoresis in 1
TAE (Tris acetate EDTA) buffer stained with ethidium bromide
and visualized under UV light. Gel photographs were scanned
through a Gel Doc System (UVitech, USA) and the amplica-
tion product sizes were evaluated using the Software Quantity
One (Bio Rad, USA).
NAB/NA + NB (Nei and Li, 1979). Where, NAB is the number
of amplied products common to both A and B. NA and NB
correspond to the number of amplied products in A and B
respectively. The resulting similarity coefcients were em-
ployed to assess the relationship among ginger accessions.
All amplied proles 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 ma-
trix. These data were used to construct dendogram for cluster
analysis based on unweighted pair group method with
arithmatic means (UPGMA) using Winbbot (IRRI, Los Ba-
nos, Philippines) (Yap and Nelson, 1996).

2.2.3. Data analysis 3. Results and discussion

Data analysis was done using Alpha Imager EC software. For
each accession the amplied fragment/band was treated as a
unit character. Amplied 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 amplied DNA fragments was esti-
mated by comparison with the molecular weight marker
100bp1500pb DNA Ladder (Bio-Basic. Inc.). Pair-wise com-
parisons of all ginger accessions based on absence or presence
of unique and shared DNA bands were used to make similarity
coefcients. Estimates of genetic similarity (S) were calculated
between all pairs of ginger accessions using the formula, S = 2
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 difcult due to the presence of sec-
ondary metabolites. A modied CTAB method by Doyle and
Doyle (1990) proved to be fruitful. The modied method in-
cluded higher incubation temperature (65 C). Random ampli-
ed polymorphic DNA and related techniques require less
DNA, but purity is necessary to ensure repeatability and con-

Table 2 Optical density of DNA isolated from different accessions of Zingiber ofcinale Rosc.
Code No. Zingiber ocinale samples Optical density (k) Ratio of 260/280 nm
260 nm 280 nm
G1 Patna (Bihar) 0.094 0.051 1.84
G2 Lucknow (U.P.) 0.034 0.019 1.78
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
162 K. Ashraf et al.

dence (Welsh and McClelland, 1990; Williams et al., 1990).
The purity of DNA determined from the ratio of optical den-
sity 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 screen-
ing 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 de-
tect distinct and polymorphic amplied products within the
species. Out of twenty decamer random primers used for 12
accessions, 07 primers did not produce any amplication at
all in the initial screening while 13 primers showed amplied
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 ma-
trix 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 1001500 bp.
The maximum and minimum number of bands was produced
by the primers OPA-11(28), and OPA-06(12), respectively (Ta-
ble 3). A total number of 275 amplied 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. ofcinale by the means of 13 RAPD primers.

Table 3 Primer sequence and amplied products of 12 accessions of Zingiber ofcinale Rosc.
Primer Sequence of primer (50 -30 ) Size of product amplied No. of amplied No. Polymorphic % Polymorphism
(bps) 1001500 bps band band
OPAA -01 AGACGGCTCC 2151181 21 20 95.23
OPA A-02 GAGACCAGAC 2581153 21 19 90.47
OPA A-03 TTAGCGCCCC 135844 14 13 92.87
OPAA -04 AGGACTGCTC 1631300 25 24 96
OPAA -05 GGCTTTAGCC 2531375 26 26 100
OPAA -06 TCAAGCTAAC 314766 12 11 91.66
OPAA -07 CTACGCTCAC 2501411 26 24 92.30
OPAA -08 TCCGCAGTAG 1451125 26 26 100
OPAA -09 AGATGGGCAG 2871400 21 21 100
OPAA -10 TGGTCGGGTG 4281232 19 17 89.47
OPAA -11 ACCCGACCTG 2921269 28 27 96.42
OPAA-12 GGACCTCTTG 280988 17 15 88.23
OPAA-15 ACGGAAGCCC 3541054 19 18 94.73
Genetic diversity analysis of Zingiber Ofcinale Roscoe by RAPD collected from subcontinent of India 163

Figure 2 RAPD amplication pattern of different accessions of Z. ofcinale using primer OPA-10, M-molecular marker (1001500 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 amplication pattern of different accessions of Z. ofcinale using primer OPA-12, M-molecular marker (1001500 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. ofcinale.

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 0.15 0.23 0.26 0.24 0.31 0.23 1
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.

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 val-
ues 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 Luc-
know 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 similar-
ity is shown by Delhi and Chandigarh with 39%. The similar-
ity 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 acces-
sions/varieties reveals the presence of strong genetic structure
between them and thus signicant differences exist in the geno-
typic diversity among themselves. Our nding is supported by
results of Mohd et al. (2004) who reported that the genetic var-
iation occurred among the three ginger cultivars from Malay-
sia 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 identication of varieties than morpho-
logical characters (Palai et al., 2007). Islam et al. (2007) nd-
ings 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 conrmatory with
the nding of Huang et al. (2003) who reported signicant
(high) genetic variations by RAPD markers in other species
at cultivar level. Our nding is also supported by Jatoi et al.
(2008) 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 ge-
netic diversity at intra specic 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 sim-
ilarity among them as compared to accessions which are far-
ther apart. This outcome is supported by Nayak et al. (2006)
who established that main cause of polymorphism could be
intraspecic variation among different cultivars. These nd-
ings will also provide an important contribution in determin-
ing resourceful management strategies for breeders for ginger
improvement program.
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