Saudi Journal of Biological Sciences (2014) 21, 159–165
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
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 Sayeed Ahmad a, Mohd. Amir a, N. Mallick a
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; RAPD; UPGMA; India
Abstract The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers 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 Ginger (Zingiber officinale 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: [email protected] (M. Mujeeb). Peer review under responsibility of King Saud University.
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2000 years (Bartley and Jacobs, 2000). It is cultivated in many tropical and subtropical countries in which, China and India are the world’s leading producers (Blumenthal et al., 2000). India recorded the highest production of ginger in the world (0.38 million tons) in 2009 (Sajeeva et al., 2011).The importance of ginger is gaining recently because of its low toxicity and its broad spectrum of biological and pharmacological applications including antitumor, antioxidant, anti-inflammatory, 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
160 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). 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 prov-
K. Ashraf et al. inces 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 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 Technology Inc., USA), 1 unit Taq DNA polymerase (Promega)
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India Table 1
List of different accessions of Zingiber officinale used in the present study.
Code No. Cultivation regions (Provinces) Latitude, Altitude G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
161
Patna (Bihar) Lucknow (U.P.) Dehradun (Utrakhand) Erode (Tamil Nadu) Bangalore (Karnataka) Nashik (Maharashtra) Trivandrum (Kerala) Delhi (Delhi) Chandigarh (Haryana) Bhopal (M.P.) Guwahati (Assam) Surat (Gujrat)
25360 39.600 N, 8580 38.400 E 26500 49.200 N, 80560 49.200 E 30180 56.5200 N, 7820 0.9600 E 11210 000 N, 77440 000 E 12580 000 N, 77340 000 E 2000 000 N, 73460 4800 E 8290 1500 N, 76570 900 E 28360 3600 N, 77130 4800 E 30450 000 N, 76460 4800 E 23150 000 N, 77250 000 E 26110 000 N, 91440 000 E 26110 000 N, 91440 000 E
Source
Date of collections
Local farmer, cultivated Herbal garden, Integral University, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer. Cultivated Local farmer, Cultivated Herbal garden, Hamdard University, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer, Cultivated Keshal nursery, Cultivated
23rd Oct. 2011 25th Oct. 2011 28th Oct. 2011 2nd Nov. 2011 4th Nov. 2011 12th Nov. 2011 7th Nov. 2011 19th Dec. 2011 17th Oct. 2011 18th Jan. 2012 22nd Nov. 2011 5th Jan. 2012
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/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 amplification 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 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 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).
2.2.3. Data analysis
3. Results and discussion
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
Table 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 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 con-
Optical density of DNA isolated from different accessions of Zingiber officinale Rosc.
Code No.
Zingiber officinale samples
Optical density (k)
Ratio of 260/280 nm
260 nm
280 nm
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
Patna (Bihar) Lucknow (U.P.) Dehradun (Utrakhand) Erode (Tamil Nadu) Bangalore (Karnataka) Nashik (Maharashtra) Trivandrum (Kerala) Delhi (Delhi) Chandigarh (Haryana) Bhopal (M.P.) Guwahati (Assam) Surat (Gujrat)
0.094 0.034 0.032 0.093 0.087 0.034 0.051 0.040 0.070 0.088 0.092 0.020
0.051 0.019 0.017 0.052 0.045 0.016 0.016 0.021 0.037 0.047 0.051 0.011
1.84 1.78 1.88 1.78 1.92 1.80 1.88 1.90 1.89 1.87 1.80 1.81
162
K. Ashraf et al.
fidence (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 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
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 (bps) 100–1500 bps
No. of amplified band
No. Polymorphic band
% Polymorphism
OPAA -01 OPA A-02 OPA A-03 OPAA -04 OPAA -05 OPAA -06 OPAA -07 OPAA -08 OPAA -09 OPAA -10 OPAA -11 OPAA-12 OPAA-15
AGACGGCTCC GAGACCAGAC TTAGCGCCCC AGGACTGCTC GGCTTTAGCC TCAAGCTAAC CTACGCTCAC TCCGCAGTAG AGATGGGCAG TGGTCGGGTG ACCCGACCTG GGACCTCTTG ACGGAAGCCC
215–1181 258–1153 135–844 163–1300 253–1375 314–766 250–1411 145–1125 287–1400 428–1232 292–1269 280–988 354–1054
21 21 14 25 26 12 26 26 21 19 28 17 19
20 19 13 24 26 11 24 26 21 17 27 15 18
95.23 90.47 92.87 96 100 91.66 92.30 100 100 89.47 96.42 88.23 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 Table 4
K. Ashraf et al. Similarity matrix table of 12 samples of Z. officinale.
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
1 0.29 0.28 0.25 0.22 0.22 0.21 0.23 0.23 0.26 0.17 0.17
1 0.28 0.27 0.23 0.21 0.22 0.21 0.19 0.23 0.21 0.2
1 0.21 0.25 0.23 0.24 0.22 0.22 0.23 0.2 0.18
1 0.29 0.26 0.32 0.15 0.22 0.2 0.15 0.2
1 0.25 0.27 0.26 0.24 0.19 0.19 0.19
1 0.27 0.23 0.22 0.21 0.19 0.15
1 0.25 0.24 0.22 0.21 0.23
1 0.39 0.3 0.22 0.26
1 0.26 0.19 0.24
1 0.23 0.31
1 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 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) 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. References Ali, B.H., Blunden, G., Tanira, M.O., Nemmar, A., 2008. Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. Food and Chemical Toxicology 46, 409–420. Bartley, J., Jacobs, A., 2000. Effects of drying on flavour compounds in Australian grown ginger (Zingiber officinale). Journal of the Science of Food and Agriculture 80, 209–215. Blumenthal, M., Goldberg, A., Brinckmann, J., 2000. Ginger root. In Herbal medicine: Expanded commission E monographs. pp. 153– 159. Connell, D.W., Sutherland, M.D., 1969. A re-examination of gingerol, shogaol and zingerone: the pungent principles of ginger (Zingiber officinale Roscoe). Australian Journal of Chemistry 22, 1033–1043. Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15. Evans, W.C., 2002. Ginger. Trease and Evans Pharmacognosy, 15th ed. WB Saunders, Edinburgh, pp. 227–280. Hamrick, J.L., Loveless, M.D., 1989. The genetic structure of tropical tree populations: associations with reproductive biology. In: Bock, J.H., Linhart, Y.B. (Eds.), Plant Evolutionary Ecology. West View Press, Boulder Colorado, pp. 131–146. Huang, H., Layne, D.R., Kubisiak, T.L., 2003. Molecular characterization of cultivated pawpaw (Asimina triloba) using RAPD markers. Journal of the American Society for Horticultural Science 128, 85–93. Islam, M.A., Meister, A., Schubert, V., Kloppstech, K., Esch, E., 2007. Genetic diversity and cytogenetic analysis in Curcuma zedoaria (Christm) Roscoe from Bangladesh. Genetic Resources and Crop Evolution 54, 149–156. Jaffery, E.H., Brown, A.F., Kurilich, A.C., Keek, A.S., Matusheski, N., Klein, B.P., 2003. Variation in content of bioactive components
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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 Sayeed Ahmad a, Mohd. Amir a, N. Mallick a
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; RAPD; UPGMA; India
Abstract The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers 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 Ginger (Zingiber officinale 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: [email protected] (M. Mujeeb). Peer review under responsibility of King Saud University.
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2000 years (Bartley and Jacobs, 2000). It is cultivated in many tropical and subtropical countries in which, China and India are the world’s leading producers (Blumenthal et al., 2000). India recorded the highest production of ginger in the world (0.38 million tons) in 2009 (Sajeeva et al., 2011).The importance of ginger is gaining recently because of its low toxicity and its broad spectrum of biological and pharmacological applications including antitumor, antioxidant, anti-inflammatory, 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
160 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). 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 prov-
K. Ashraf et al. inces 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 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 Technology Inc., USA), 1 unit Taq DNA polymerase (Promega)
Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India Table 1
List of different accessions of Zingiber officinale used in the present study.
Code No. Cultivation regions (Provinces) Latitude, Altitude G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
161
Patna (Bihar) Lucknow (U.P.) Dehradun (Utrakhand) Erode (Tamil Nadu) Bangalore (Karnataka) Nashik (Maharashtra) Trivandrum (Kerala) Delhi (Delhi) Chandigarh (Haryana) Bhopal (M.P.) Guwahati (Assam) Surat (Gujrat)
25360 39.600 N, 8580 38.400 E 26500 49.200 N, 80560 49.200 E 30180 56.5200 N, 7820 0.9600 E 11210 000 N, 77440 000 E 12580 000 N, 77340 000 E 2000 000 N, 73460 4800 E 8290 1500 N, 76570 900 E 28360 3600 N, 77130 4800 E 30450 000 N, 76460 4800 E 23150 000 N, 77250 000 E 26110 000 N, 91440 000 E 26110 000 N, 91440 000 E
Source
Date of collections
Local farmer, cultivated Herbal garden, Integral University, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer. Cultivated Local farmer, Cultivated Herbal garden, Hamdard University, Cultivated Local farmer, Cultivated Local farmer, Cultivated Local farmer, Cultivated Keshal nursery, Cultivated
23rd Oct. 2011 25th Oct. 2011 28th Oct. 2011 2nd Nov. 2011 4th Nov. 2011 12th Nov. 2011 7th Nov. 2011 19th Dec. 2011 17th Oct. 2011 18th Jan. 2012 22nd Nov. 2011 5th Jan. 2012
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/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 amplification 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 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 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).
2.2.3. Data analysis
3. Results and discussion
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
Table 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 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 con-
Optical density of DNA isolated from different accessions of Zingiber officinale Rosc.
Code No.
Zingiber officinale samples
Optical density (k)
Ratio of 260/280 nm
260 nm
280 nm
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
Patna (Bihar) Lucknow (U.P.) Dehradun (Utrakhand) Erode (Tamil Nadu) Bangalore (Karnataka) Nashik (Maharashtra) Trivandrum (Kerala) Delhi (Delhi) Chandigarh (Haryana) Bhopal (M.P.) Guwahati (Assam) Surat (Gujrat)
0.094 0.034 0.032 0.093 0.087 0.034 0.051 0.040 0.070 0.088 0.092 0.020
0.051 0.019 0.017 0.052 0.045 0.016 0.016 0.021 0.037 0.047 0.051 0.011
1.84 1.78 1.88 1.78 1.92 1.80 1.88 1.90 1.89 1.87 1.80 1.81
162
K. Ashraf et al.
fidence (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 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
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 (bps) 100–1500 bps
No. of amplified band
No. Polymorphic band
% Polymorphism
OPAA -01 OPA A-02 OPA A-03 OPAA -04 OPAA -05 OPAA -06 OPAA -07 OPAA -08 OPAA -09 OPAA -10 OPAA -11 OPAA-12 OPAA-15
AGACGGCTCC GAGACCAGAC TTAGCGCCCC AGGACTGCTC GGCTTTAGCC TCAAGCTAAC CTACGCTCAC TCCGCAGTAG AGATGGGCAG TGGTCGGGTG ACCCGACCTG GGACCTCTTG ACGGAAGCCC
215–1181 258–1153 135–844 163–1300 253–1375 314–766 250–1411 145–1125 287–1400 428–1232 292–1269 280–988 354–1054
21 21 14 25 26 12 26 26 21 19 28 17 19
20 19 13 24 26 11 24 26 21 17 27 15 18
95.23 90.47 92.87 96 100 91.66 92.30 100 100 89.47 96.42 88.23 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 Table 4
K. Ashraf et al. Similarity matrix table of 12 samples of Z. officinale.
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
1 0.29 0.28 0.25 0.22 0.22 0.21 0.23 0.23 0.26 0.17 0.17
1 0.28 0.27 0.23 0.21 0.22 0.21 0.19 0.23 0.21 0.2
1 0.21 0.25 0.23 0.24 0.22 0.22 0.23 0.2 0.18
1 0.29 0.26 0.32 0.15 0.22 0.2 0.15 0.2
1 0.25 0.27 0.26 0.24 0.19 0.19 0.19
1 0.27 0.23 0.22 0.21 0.19 0.15
1 0.25 0.24 0.22 0.21 0.23
1 0.39 0.3 0.22 0.26
1 0.26 0.19 0.24
1 0.23 0.31
1 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 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) 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
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