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: [email protected] (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 K. Ashraf et al.
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 Till date several studies have been reported on genetic
compounds such as gingerol, shogaol, ginger protease, diversity study of different ginger varieties in India but to our
capsaicin and several sesquiterpenes like zingiberol, knowledge very less or no study has ever been reported on
zingiberenol and these constituents may vary depending on the diversity collected from all major ecological zones or provinces
place of origin and whether the rhizomes are fresh or dry (Tang of India. Since ginger is a very poorly studied crop and its
and Eisenbrand, 1992; Ali et al., 2008). Over 50 components of molecular information is limited, hence it is imperative to know
the oil present in ginger are mainly monoterpenoids [b- the genetic diversity among different accessions from Indian
phellandrene, (+)-camphene, cineole, geraniol etc.] and subcontinent.
sesquiterpenoids [a zingiberene (30–70%), b-
sesquiphellandrene (15–20%), bbisabolene (10–15%), (E-E)-a-
farnesene, arcurcumene, and zingiberol] (Langner et al., 1998; 2. Materials and methods
Evans, 2002).The phenolic ketone compounds such as 6-
gingerol, 8-gingerol and 10-gingerol are the principle active 2.1. Plant material
pungent compounds (Connell and Sutherland, 1969). The
pungency of fresh ginger is primarily due to the gingerols, The crude drug samples of rhizomes of Z. officinale were
which are a homologous series of phenols. The pungency of dry collected in the months of October (2011) – January (2012) from
ginger mainly results from shogaols which are the dehydrated twelve different regions (Table 1) of India. All collected samples
forms of gingerols. Shogaols are formed from the were authenticated by the taxonomist Professor M.P. Sharma,
corresponding gingerol during thermal processing (Wohlmuth Department of Botany, Faculty of Science, Jamia Hamdard, New
et al., 2005). Ginger protease plays a very important role in Delhi. All voucher specimens were deposited in the Molecular
meat tenderization (10-fold activity) and it can significantly Ecology Laboratory, Jamia Hamdard, New Delhi, India.
improve the flavor as well as quality of the meat by increasing
nutritious value (Naveena and Mendiratta, 2001; Zhou et al., 2.2. Genetic diversity analysis
1996). Ginger protease can also improve the quality of food
processing without reducing the nutritious value. It also 2.2.1. Isolation of genomic DNA
separates casien protein into smaller peptide (Song et al., 2001). Conventional DNA isolation protocol suggests the use of leaves
It is reported that variations in quantity and quality of the which are devoid of secondary metabolite and this metabolite
polyphenols present in plant foods occurred due to different causes hindrance in DNA isolation and makes the whole process
factors, such as plant genetics and cultivar, soil composition tedious. So we developed CTAB modified method. In brief,
and growing conditions, maturity state, and post harvest genomic DNA of frozen rhizome samples was isolated by
conditions (Jaffery et al., 2003). Besides this, most of the crop modified cetyl trimethyl ammonium bromide (CTAB) extraction
improvement programs of ginger are restricted to the method (Doyle and Doyle, 1990). 1 g of rhizome sample along
assessment and selection of naturally occurring clonal with polyvinylpyrrolidone was triturated in liquid nitrogen to fine
variations (Rout et al., 1998; Palai and Rout, 2007).Therefore, powder. 5 ml of 1% CTAB (100 mM Tris–HCl buffer pH 8.0, 1.4
diversity analysis and identification of genetically distant clones M NaCl, 20 mM, EDTA, 1% mercaptoethanol) buffer was added
or genotypes are vital to the ginger improvement program. to the homogenate, and centrifuged at 10,000 rpm for about 15
The assessment of genetic diversity may be done within and min. Added, 2 vol. of 2% CTAB (100 mM Tris–HCl buffer, 1.4
between populations at molecular level by using various M NaCl, 20 mM
techniques like allozymes or DNA analysis (Mondini et al., EDTA) to the collected aqueous phase. This mixture was
2009). During the past decades, use of molecular markers is incubated at 65 C for about 60 min with intermittent shaking. The
gaining attention to reveal polymorphisms at the DNA level. suspension was then cooled to room temperature and equal
Different marker based techniques are available for the volume of chloroform and isoamyl alcohol (24:1) was added. The
identification of plants. Out of these, molecular marker based is mixture was then centrifuged at 10,000 rpm for 15 min. The
more accepted because it overcomes many of the limitations of aqueous phase was collected, and to it was added 0.6 volume of
morphological and biochemical techniques since they are not cold isopropanol and 1/30 volume of sodium acetate (3 M, pH
affected by the environmental or developmental stage and can 5.2) and incubated at 20 C for 1 h. The sample was centrifuged at
detect a variation at the DNA level (Tingey and Tufo, 1993). 10,000 rpm for 15 min to obtain the DNA pellet. The pellet was
PCR-based molecular markers were extensively used in many washed with 80% ethanol twice, air dried and dissolved in TE
plant species for identification, phylogenetic analysis, buffer (10 mM Tris buffer, pH 8.0, 1 mM Na 2 EDTA). The
population studies and genetic linkage mapping. RAPD isolated DNA was treated with RNase A (10 lg/ml) at 37 C for 30
markers are markers of choice, because of its simplicity and min. DNA concentration and purity were determined by
low-cost nature, rapid, inexpensive and effective system for measuring the absorbance of diluted DNA solution at 260 nm and
studying plant genetic relationships (Williams et al., 1990). The 280 nm. The quality of the DNA was determined using agarose
RAPD markers could also be used in the study of genetic gel electrophoresis stained with ethidium bromide.
variability of species or natural populations (Lashermes et al.,
1993; Wilkie et al., 1993) and in the study of genotype 2.2.2. RAPD amplification
identification (Wilde et al., 1992; Koller et al., 1993; Wolff and
Polymerase chain reaction (PCR) was performed according to the
Peters-Van Run, 1993).
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 arithmatic means (UPGMA) using Winbbot (IRRI, Los Ban˜ os,
performed in a thermal cycler (Master Cycler, Eppendorf, USA) Philippines) (Yap and Nelson, 1996).
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 3. Results and discussion
C for 5 min. Amplification products were separated alongside a
molecular weight marker (100 bp plus ladder, 3.1. Genetic diversity analysis
M/SBangaloreGenei)by1.2%agarosegelelectrophoresisin1·
TAE(TrisacetateEDTA)bufferstainedwithethidiumbromide and Genetic variability studies in ginger collected from different
visualized under UV light. Gel photographs were scanned through geographical regions of India have been carried out using
a Gel Doc System (UVitech, USA) and the amplification product RAPD markers. DNA was isolated by CTAB method. DNA
sizes were evaluated using the Software Quantity One (Bio Rad, extraction of ginger proved difficult due to the presence of
USA). secondary metabolites. A modified CTAB method by Doyle
and Doyle (1990) proved to be fruitful. The modified method
2.2.3. Data analysis included higher incubation temperature (65 C). Random
Data analysis was done using Alpha Imager EC software. For amplified polymorphic DNA and related techniques require
each accession the amplified fragment/band was treated as a unit less DNA, but purity is necessary to ensure repeatability and
character. Amplified agarose gel pictures were compared with confidence (Welsh and McClelland, 1990; Williams et al.,
each other and data were scored as the absence (0) or presence (1) 1990). The purity of DNA determined from the ratio of optical
of a DNA band for each of the primer-accession combination. The density of 260/280 ratio which ranged from 1.78 to 1.92 for the
size of amplified DNA fragments was estimated by comparison samples indicates the purity of DNA in all samples (Table 2).
with the molecular weight marker 100bp–1500pb DNA Ladder The present study offers an optimization of primer screening
(Bio-Basic. Inc.). Pair-wise comparisons of all ginger accessions for evaluation of genetic relationship among the twelve
based on absence or presence of unique and shared DNA bands accessions of ginger through RAPD analysis. Twenty decamer
were used to make similarity coefficients. Estimates of genetic primers from Operon Technologies (Alameda, California,
similarity (S) were calculated between all pairs of ginger USA) were initially screened using one species of Delhi to
accessions using the formula, S = 2 N AB/NA + NB (Nei and Li, determine the suitability of each primer for the study. Primers
1979). Where, NAB is the number of amplified products common were selected for further analysis based on their ability to detect
to both A and B. NA and NB correspond to the number of amplified distinct and polymorphic amplified products within the species.
products in A and B respectively. The resulting similarity Out of twenty decamer random primers used for 12 accessions,
coefficients were employed to assess the relationship among 07 primers did not produce any amplification at all in the initial
ginger accessions. All amplified profiles were analyzed together screening while 13 primers showed amplified polymorphic
162 K. Ashraf et al.
pattern. These primers were then used for RAPD analysis for 12 maximum and minimum number of bands was produced by the
accessions. The commercial software package NTSYS-PC primers OPA-11(28), and OPA-06(12), respectively (Table 3).
(Rohlf, 1995) was used to develop similarity matrix These data A total number of 275 amplified fragments was scored across
were then used for constructing dendrogram (Fig. 1) for cluster twelve accessions of ginger for the selected primers, and was
analysis based on Unweighed pair group method with used to estimate genetic relationships among themselves. Out of
arithmatic mean (UPGMA). The selected primers generated 275 fragments obtained, 261 fragments (94.90%) were
distinctive products in the range of 100–1500 bp. The

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 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, G11Guwahati
and G12-Surat.
polymorphic. The pattern of RAPD produced by the primers that there occurs a high degree of genetic variation in ginger
OPA-10 and OPA-12 are shown in (Figs. 2 and 3). collected from the Asian regions.
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 4. Conclusions
indicated that these accessions were distantly related to each other
as reported by (Palai and Rout, 2007). Based on the dendrogram, The main emphasis of the present study was to assess the
the 12 accessions were grouped into two main clusters (cluster I genetic diversity at intra specific level among the 12 accessions
and cluster II). Cluster I is divided into sub cluster IA and IB. of ginger of Indian subcontinent using RAPD markers. RAPD
These subcluster contain Patna and Lucknow with similarity of analysis shows that there is a high level of polymorphism
29%. Dehradun is similar with Patna and Lucknow with 28%. In among different accessions. From this study, it was understood
cluster IB, Erode and Trivandrum shows similarity of 32%. In that each location varied with respect to environmental factors
lower cluster II, maximum similarity is shown by Delhi and and genetic parameters. Results showed that the accessions
Chandigarh with 39%. The similarity between Bhopal and Surat is whose cultivation regions are very close shows maximum
found to be 31%. Three pairs of ginger varieties show least similarity among them as compared to accessions which are
similarity among all accessions of Erode and Guwahati, Erode farther apart. This outcome is supported by Nayak et al. (2006)
and Delhi, and Nashik and Surat at 15%. The high difference in who established that main cause of polymorphism could be
gene diversity among accessions/varieties reveals the presence of intraspecific variation among different cultivars. These findings
strong genetic structure between them and thus significant will also provide an important contribution in determining
differences exist in the genotypic diversity among themselves. resourceful management strategies for breeders for ginger
Our finding is supported by results of Mohd et al. (2004) who improvement program.
reported that the genetic variation occurred among the three
ginger cultivars from Malaysia using a RAPD marker. RAPD
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