Comparative Study of RAPD and ISSR Markers to Assess the Genetic Diversity of Betel Vine (Piper betle L.) in Orissa, India
Aparna Priyadarshini Patra,
Arup Kumar Mukherjee
In Orissa Betel vine (Piper betle L., family Piperaceae) is an important asexually propagated cash crop comprising of several cultivars. There are many cultivares but they are not well demarcated due to similarities in the morphological characters and in certain places same cultivars are cultivated under different local name. Therefore, in the present study DNA fingerprinting technique has been used to differentiate cultivars of betel vine for crop improvement programme. So Comparative study of both RAPD and ISSR markers analysis were used to establish genetic identities and evaluate genetic diversity among fifteen cultivars of betel vine grown in different parts of Orissa. Thirty RAPD and 25 ISSR primers were tested to resolve the genetic diversity among the cultivars. Twenty RAPD and 18 ISSR primers resulted in 523 amplicons. Out of these 504 were polymorphic loci and 54 were found to be unique. The extent of genetic diversity and relatedness among 15 cultivars were computed through Jaccards similarity coefficient. Maximum similarity (0.68) was observed between Balipana and Birkoli and minimum (0.114) for Banglamandesore chitalpudi and Halisahar Sanchi. All the cultivars were related with each other with an average similarity of 0.2913. Dendrogram showed Godibangala was separated from rest of the species into isolated clade in both the analysis. Correlation between RAPD and ISSR marker was very low (r = 0.17). RAPD showed high correlation with all the primers.
Received: July 25, 2010;
Accepted: January 14, 2011;
Published: February 07, 2011
Betel vine (Piper betle L., family Piperaceae) is an important, traditional
and ancient crop of India. Leaves of betel vine have been used with condiments
such as areca nut, kattha, cloves, cardamom, fennel and candid rose for chewing
purpose. It has also been used in the Indian system of medicine (Rawat
et al., 1989a; Garg and Rajshree, 1992; Sandhya
et al., 1995) for digestive, caraminative, stimulant, antiseptic
and antifungal purposes. A phenolic compound, hydroxy-chavicol, with anticarcinogenic
property has also been identified in betel leaves (Bhide
et al., 1991). Fresh juice of betel leaves is also used in many ayurvedic
preparations (Sharma, 1991).
Betel vine is widely cultivated in the states of Uttarpradesh, Bihar, Madhyapradesh,
Northeastern India, Maharashtra, Karnataka, West Bengal, Orissa andhra Pradesh,
Tamilnadu, Kerala and Andamans in India. Betel vines are dioecious in nature.
Under controlled hybridization, attempts have been made to cross different landraces
and in some of these experiments, viable seed set has been reported (Maiti
and Shivashankar, 2002). However, as a crop, propagation is only through
vegetative means. Its cultivation in northern India under sub-tropical conditions
has been shown to be a unique case of plant establishment under anthropogenically
regulated microclimatic conditions (Kumar, 1999).
The betel vine growers invariably named their cultivars with local or vernacular
names. Therefore, these cultivated betel vines are nothing but landraces. A
survey over several years indicated that there are many local cultivars (landraces)
of betel vines in Orissa, India. Many of these land races differ from each other
in organoleptic properties (Verma et al., 2004).
Scrutiny over the landrace names and their etymology, suggests that a given
landrace may be named differently in different regions and more than one land
race may have same name. On the basis of chemical constituents and essential
oils, five prominent groups of betel vine landraces, namely, Bangla, Kapoori,
Metha, Sanchii and Desavari have been recognized (Rawat
et al., 1989b). The research work on genetic variation among the
landraces using molecular or biochemical methods is however, scanty. In the
absence of any systematic attempts to resolve this nomenclature problem and
since betelvines are vegetatively propagated, most of these names are as ancient
as the cultivation of betelvine itself. A few isolated efforts have been made
to rationalize the different landraces and to identify similar or dissimilar
types among them. No studies have been reported on the phylogeny of betelvine
in Orissa. So this report will be the first to disclose the phylogenetic relationship
among the betelvine cultivars. The evaluation of genetic diversity and phylogenetic
relationship among the cultivars would promote the efficient use of genetic
variations in the breeding programme (Paterson et al.,
1991). As it is a cash crop of coastal Orissa, many people depend on it
for their livelihood. This study will give a better idea about the distribution
and cropping pattern of Betel vine.
The PCR based method for DNA profiling, Random Amplified Polymorphic DNA (RAPD)
(Welsh and Mc Clelland, 1990; Williams
et al., 1990) and inter simple sequence repeats (ISSR) (Bornet
and Branchard, 2001; Zietkiewicz et al., 1994)
were used to identify the duplicates or sort the germplasm and to estimate genetic
diversity among the plants (Virk et al., 1995).
This technique was used in our laboratory to determine genetic variation in
intra specific levels (Ranade et al., 1997; Farooqui
et al., 1998; Goswami and Ranade, 1999).
In the present study we show the application of RAPD (Lynch
and Milligan, 1994; Tembe and Deodhar, 2010; Tertivanidis
et al., 2008) and ISSR (Gantait et al., 2010;
Fares et al., 2009) technique in assessing the
diversity amongst the betel vine landraces collected from different parts of
Orissa and maintained under All India Coordinated Research Project (AICRP) on
betel vine, Bhubaneswar, Orissa, India. Although, RAPD (Colagar
et al., 2010) and ISSR profiling differ mainly in the pattern of
fragment they amplify, the data interpretation is identical and can be combined
for statistical analysis (Souframanien and Gopalakrishna,
2004). ISSRs are semiarbitrary markers amplified by PCR in the presence
of one primer complementary to a target microsatellite. Like RAPDs, ISSRs markers
are also quick and easy to handle and have been successfully utilized for the
MATERIALS AND METHODS
Sample collection: Betel vine landraces were collected and maintained
under All India Coordinated Research Project (AICRP), OUAT, Bhubaneswar on Betel
vine (Table 1). The study was conducted during the period
of 2005-06. Young leaf tissue was harvested from field grownvines, washed properly
to remove dirt, mopped dry and quickly frozen.
|| Name, Acc No. and source of collection of germ plasm of betel
Then the frozen leaves were powered using liquid nitrogen. The powders were
either used for isolation of DNA immediately or were stored in a deep freezer
(-80°C) for long term storage.
Isolation of genomic DNA: The total genomic DNA was isolated from powdered
and young leaf tissue of betel vine landraces by using modified CTAB method
(Doyle and Doyle, 1990). Three independent DNA preparations
were made from leaf tissue collected from each land races. The quantity and
quality of DNA samples were estimated by comparing band intensities on agarose
gel (Ranade et al., 2002).
RAPD marker analysis: RAPD amplification was performed with random decamer primers obtained from Operon Technologies (Alameda, CA, USA). Thirty arbitrary RAPD primers were tested for PCR amplification. Twenty of them were chosen for the analysis because they produced highly readable and reproducible bands (Table 2). RAPD analysis was done by using primers from A, B, D, H and N series (Table 2) were produced highly reproducible bands. The experiment was standardized using various primers, template DNA and Mg2+ concentration to determine the optimum result. The final amplification reactions contained Each reaction mixture of 25 μL contained 20 ng template DNA, 2.5 μL of 10 X assay buffer (100 mM Tris HCl pH 8.3, 500 mM KCl and 0.1% gelatin), 1.5 mM MgCl2, 200 mM each dNTPs, 15 ng primer and 0.5 U Taq DNA polymerase (Banglore genei, Banglore). The reaction was cycled 42 times at 94°C for 1 min, 37°C for 1 min and 72°C for 2 min in a thermo cycler (Applied Biosystem, Model 9700). The final extension cycle allowed an additional incubation for 7 min at 72°C.
ISSR marker analysis: For ISSR amplification some anchored and non anchored
microsatellite primers designed in our laboratory were randomly selected and
used. Twenty-five arbitrary ISSR primers were used for PCR amplification. Eighteen
of them were chosen for the analysis because they produced highly readable and
reproducible bands (Table 3). Each reaction mixture of 25
μL contained 20 ng template DNA, 2.5 μL of 10 X assay buffer (100
mM Tris HCl pH 8.3, 500 mM KCL and 0.1% gelatin), 1.5 mM MgCl2, 200
mM each dNTPs, 15 ng primer and 0.5 U Taq DNA polymerase (Bangalore genei, Bangalore).
|| Details of RAPD primers analysis
|aPrimers are from operon technology
||Details of ISSR primers analysis
|aPrimers are from operon technology
The amplification was carried out in a thermal cycler (Applied Biosystem, Model
9700). The reaction was cycled 42 times at 94°C for 1 min, 45-55°C for
1 min for annealing and 72°C for 2 min in a thermo cycler. The final extension
cycle allowed an additional incubation for 7 min at 72°C for complete polymerization.
Agarose gel electrophoresis: The amplified products were separated by
electrophoresis through 1.5% agarose gel in 1X TAE buffer pH 8 (Sambrook
et al., 1989) visualized and photographed using gel documentation
system (Bio rad USA) after staining with ethidium bromide.
Band profile reproducibility: Three replicate DNA extractions from leaves of Piper betel were used to assess the consistency of the band profiles. RAPD and ISSR amplifications were repeated at least three times and only the reproducible PCR products were scored.
Data analysis: Data (fragment size of all the amplification products estimated from gel by comparison with standard molecular weight marker, 1 kbp DNA ladder) were scored as discrete variables using 1 to indicate presence and 0 to indicate absence of a band. A pair wise matrix of distance between landraces was determined for the cumulative RAPD and ISSR data.
Jaccards coefficient of similarity (Jaccard, 1901)
was measured and a phylogram based on similarity coefficients generated by Unweighted
Pair Group Method using Arithmetic average (UPGMA) (Sneath and
Sokal, 1973) and sequential agglomerative hierarical nested clustering (SHAN)
was obtained. Most informative primers were obtained by comparing all primers
with that of pooled data using Mantel Z statistics (Mantel,
1967). The entire analysis was performed using the statistical package NYTSY
2.0 e. Principal Co ordinate Analysis (PCA) was used to retrieve information
about the clustering pattern of analyzed the primers (Semagan
et al., 2000).
RESULTS AND DISCUSSION
The betelvine DNAs were tested in RAPD and ISSR reactions in triplicate. The initial pilot reactions were carried out to determine the optimum primer, template and Mg2+ concentrations (data not shown). Subsequently, the entire set of betel vine DNAs were tested with thirty decamer primers for RAPD. Out of these twenty RAPD primers produced highly reproducible and scorable bands (Table 2) were chosen for analysis. The profiles were considered consistent if at least two of the three DNA preparations revealed identically sized prominent bands after amplification with a given primer.
The highest number of fragments (16) was amplified by the primer OPA18 (Fig.
1), OPD20 and OPN16 and that were lowest (10) by primer OPD03. The primers
OPA18 (Fig. 1), OPD20 and OPN16 (Fig. 2)
produced maximum number of polymorphic bands (16) and minimum (9) in case of
OPD03. The OPD03 and OPD18 produced single monomorphic band. Resolving power
was highest for primer OPD20 and OPN16 (12.667) and lowest for OPD02 (5.38)
(Table 2). Primer index was highest in OPA17 (7.85) and lowest
in case of OPD18 (2.824) (Table 2).
|| Banding pattern of OPA18 on 1.5% agarose gel
|| Banding pattern of OPN16 on 1.5% agarose gel
|| Banding pattern of (GACA)4 on 1.5% agarose gel
Jaccards similarity coefficient showed landrace Balipana and Birkoli
were closely related having similarity value 0.779 and landrace Andaman Local
and Birkoli were widely apart with similarity of 0.119.
Twenty-five random oligonucleotide primers were tested for ISSR. Out of these eighteen ISSR primers produced highly reproducible and scorable bands (Table 3) were chosen for analysis. Resolving power and primer index for ISSR were nearly same as comparison to RAPD marker. Maximum numbers of bands (16) were resolved for the primer (GACA)4 and lowest numbers of bands (10) were resolved for (GTG)5. The highest number of polymorphic loci were detected by primer (GACA)4 (Fig. 3) and lowest by (GTG)5. Resolving power of primer (AGG)6 was maximum 8.933 and in case (GTG)5 it was lowest 3.867 (Table 3). Maximum Primer index was 4.75 in case of (GA)9T and minimum in case of (GTG)5 2.75 (Table 3).
When both markers were combined together it was observed that the average similarity between two cultivars was 0.2913. Highest similarity observed between the cultivars Birkoli and Balipana (0.68). Lowest similarity observed between Halisahar Sanchi and Bagla Mandasore Chitalpudi (0.114). From the above analysis RAPD maker shows Godibangala landrace was clustered into separate clade (Fig. 4). In ISSR analysis Godibangla andaman Local and Bagla Mandasore Chitalpudi are the subclade of one main clade (Fig. 5). Both RAPD and ISSR (Fig. 6) showed similar results as RAPD showing Godibangala as separate clade. The clustering of 15 cultivars was confirmed using Principal Co ordinate Analysis (PRINCORDA) which revealed the similar result obtained from SHAN clustering (Fig. 7). The correlation between RAPD and ISSR markers obtained using Mentel Z correlation analysis was very low (Fig. 8).
|| Cluster analysis of cumulative RAPD data for betel vine land
|| Cluster analysis of cumulative ISSR data for betel vine land
|| Cluster analysis of cumulative RAPD and ISSR data for betel
vine land race
|| Principal Co ordinate analysis of betel vine varieties
|| Correlation analysis of combined RAPD and ISSR markers (R
Betel vine is a very important medicinal and cash crop in Orissa as well as
in India. It is one of the heritage crops of India. Improvement of this crop
is primarily based on recombination breeding programme that utilizes superior
landraces possessing resistance to biotic and abiotic stress, taken as parents.
The different land races were distinguished earlier on the basis of the leaf
essential oils (Rawat et al., 1989b). However,
the extent of variation among and between them is not easily analysed due to
its vegetative propagation attributes. Under these conditions, RAPD technique
could reveal within-landrace type variation more efficiently (Verma
et al., 2004). Since, success of the improvement programme is primarily
dependent on choice of genetically diverse parents. So an attempt has been taken
in this study to assess the genetic diversity of 15 cultivars at molecular level
for identification of superior parents for the purpose of recombination breeding.
RAPD and ISSR markers were used to characterize and compare the genetic diversity
among the Betel vine cultivars. Both RAPD and ISSR showed similar result. But
the ISSR technique generated more bands per primer and revealed higher levels
of polymorphism, so we recommend ISSR for future studies. RAPD and ISSR are
PCR based arbitrary oligonucleotides primers which generally used to amplify
the complementary sequence in the genome. So it can be easily used without prior
knowledge of the genome sequence. On an average 13.78 bands have been amplified
by each primer which is reasonably good (Ranade et al.,
Similarity value showed that Bali pana and Birkoli both are from Orissa are the closest cultivars. Similarly Bangla Mandasore Chitalpudi from Andhra Pradesh and Halisahar Sanchi from West Bengal are most distantly related. Other varieties like Awanipan and Gandhi pan shared a same node interestingly both of them were collected from Assam (Table 1). Maghai and Ramtake Bangala shared same node and both of them were collected from Madhya Pradesh (Table 1) which indicates that there are certain relationship between geographical distribution and genetic diversity.
However, there is no relationship among the cultivars if response to different
diseases were considered. In fact it was expected that DNA fragments from the
entire genomic DNA amplified, only one or few genes control the disease reactions.
The high boot strap value of different cultivars in the present investigation
indicated that the major clustering pattern wont change even if some other
markers are added (Nybom, 2004). The different groups
like Bangla, Sanchi and others could not be separated out which contradicted
the earlier report of Rawat et al. (1989a), Ranade
et al. (2002) and Verma et al. (2004).
When both the markers are compared, it was observed that RAPD shows high correlation
with that of combined markers. Surprisingly, the betel vines, despite being
vegetatively propagated crop showed considerably less similarity than expected
or as reported for other vegetatively propagated crop (Breto
et al., 2001; Vega et al., 2001).
Lowest levels of polymorphism (0.8%) detected for a plant species by RAPD analysis,
for Agave tequilana var azul plants (Vega
et al., 2001). However, the betelvines exhibit less variability in
morphological characters compared to other vegetatively propagated plant. The
betelvines, though vegetatively propagated, differ considerably from the agaves
by exhibiting greater diversity amongst the landraces. It is thus possible that
centuries of cultivation by vegetative means have fixed the differences among
the groups of landraces. Alternatively, the different landraces may have been
derived from several ancestral and diverged founders or seed derived plants
before intensive vegetative cultivation practices fixed the variations by eliminating
selection of the plants on the basis of sexual reproduction (Verma
et al., 2004). This could be another reason for the greater than
expected diversity among the landraces and groups. The RAPD and ISSR profiles,
however, revealed relative variability among the betelvine landraces. Clearly
there is scope for large-scale application of RAPD (Bussell,
1999) for analysis of obligate vegetatively propagated plants.
One interesting result from the present study suggests the landraces collected from different geographical region and climatic condition were clustered into one clade. Godibangla found in a separate clade with all the primers it may genetically different from other lanraces. So further indepth study required revealing this difference.
In the betelvine, DNA methodologies have become a clear and powerful impact
on understanding the origin, evolution and genome relationships among the plant
species. However the routine use of molecular DNA marker for identification
of plant collection might be very important to get more and better understanding
about the genome relationship of the related plant species. Therefore, more
studies are planned on chromosome complements and genomes to clarify and justify
the species taxonomical relationships and evolution of the species. We are also
planning to extend present studies by using RFLP, AFLP and microsatellite marker
on the other congneric species in betelvine distributed in India also to use
those molecular markers for studies of genetic differentiation among the wild
species to identify areas of maximum diversity and to estimate genetic variability
in natural population.
Authors are thankful to Dr. S.K. Mishra, Project Co-ordinator of All India Co-ordinated Research Project on Betel vine, centre at Bhubaneswar under ICAR for supplying plant materials. We are also grateful to Dr. Dayanidhi Mahapatra, Prof and Head Dept. of Agricultural Biotechnology, OUAT for providing necessary facility to carry out experiments.
Breto, M.P., C. Ruiz, J.A. Pina and M.J. Asins, 2001.
The diversification of Citrus clementina
Hort. ex Tan., a vegetatively propagated crop species. Mol. Phylogenet. Evol., 21: 285-293.CrossRef | PubMed | Direct Link |
The distribution of random amplified polymorphic DNA (RAPD) diversity amongst populations of Isotoma petraea
(Lobeliaceae). Mol. Ecol., 8: 775-789.CrossRef | Direct Link |
Bhide, S.V., M.B.A. Zariwala, A.J. Amonlar and M.A. Azuine, 1991.
Chemopreventive efficacy of a betel leaf extract against benzo[a]pyrene-induced forestomach tumors in mice. J. Ethnopharmacol., 34: 207-213.CrossRef | PubMed | Direct Link |
Bornet, B. and M. Branchard, 2001.
Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting. Plant Mol. Biol. Rep., 19: 209-215.Direct Link |
Colagar, A.H., M. Saadati, M. Zarea and S.A. Talei, 2010.
Genetic variation of the iranian Sclerotinia sclerotiorum
isolates by standardizing DNA polymorphic fragments. Biotechnology, 9: 67-72.CrossRef | Direct Link |
Doyle, J.J. and J.L. Doyle, 1990.
Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.Direct Link |
Fares, K., F. Guasmi, L. Touil, T. Triki and A. Ferchichi, 2009.
Genetic diversity of pistachio tree using inter-simple sequence repeat markers ISSR supported by morphological and chemical markers. Biotechnology, 8: 24-34.CrossRef | Direct Link |
Farooqui, N., S.A. Ranade and P.V. Sane, 1998.
RAPD profile variation amongst provenances of neem. Biochem. Mol. Biol. Int., 45: 931-939.PubMed | Direct Link |
Gantait, S., N. Mandal, S. Bhattacharyya and P.K. Das, 2010.
Determination of genetic integrity in long-term micropropagated plantlets of Allium ampeloprasum
L. using ISSR markers. Biotechnology, 9: 218-223.CrossRef | Direct Link |
Garg, S.C. and R. Jain, 1992.
Biological activity of the essential oil of Piper betle
L. J. Essent. Oil Res., 4: 601-606.CrossRef | Direct Link |
Goswami, M. and S.A. Ranade, 1999.
Analysis of variations in RAPD profiles among accessions of prosopis. J. Genet., 78: 141-147.Direct Link |
Jaccard, P., 1901.
Etude comparative de la distribution florale dans une portion des Alpes et dejura. Bull. Soc. Vaudoise Sci. Nat., 37: 547-579.
Kumar, N., 1999.
Betelvine (Piper betle
L.) cultivation: A unique case of plant establishment under anthropogenically regulated microclimatic conditions. Indian J. Hist. Sci., 34: 19-32.Direct Link |
Lynch, M. and B.G. Milligan, 1994.
Analysis of population genetic structure with RAPD markers. Mol. Ecol., 3: 91-99.CrossRef | PubMed | Direct Link |
Maiti, S. and K.S. Shivasankara, 2002.
Technical Bulletin, Genetic Resources in Betelvine in India. (All India Co-ordinate Research Project on Betel Vine. National Research Centre for Medicinal and Aromatic Plant, Anand, Gujrat
Mantel, N., 1967.
The detection of disease clustering and a generalized regression approach. Cancer Res., 27: 209-220.Direct Link |
Nybom, H., 2004.
Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol. Ecol., 13: 1143-1155.CrossRef | PubMed | Direct Link |
Paterson, A.H., S.D. Tanksley and M.E. Sorrells, 1991.
DNA markers in plant improvement. Adv. Agron., 46: 39-90.CrossRef | Direct Link |
Ranade, S.A., A. Verma, M. Gupta and N. Kumar, 2002.
RAPD profile analysis of betel vine cultivars. Biol. Planta., 45: 523-527.CrossRef | Direct Link |
Rawat, A.K.S., R. Bannerjee and V.R. Balasubrahmanyam, 1989.
Chemical polymorphism of essential oil of Piper betle
L. grown in India. Feddes Rep., 100: 331-334.
Rawat, A.K.S., R.D. Tripathy, A.J. Khan and V.R. Balasubrahmanyam, 1989.
Essential oil components as markers for identification of Piper betle
L. cultivars. Biochem. Syst. Ecol., 17: 35-38.CrossRef | Direct Link |
Prabhu, M.S., K. Patel, G. Sarawathi and K. Srinivasan, 1995.
Effect of orally administered betel leaf (Piper betle
Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats. Indian J. Exp. Biol., 33: 752-756.PubMed | Direct Link |
Sambrook, J., E.F. Fritsch and T. Maniatis, 1989.
Molecular Cloning: A Laboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory, New York
Semagan, K., A. Bjornstad, B. Stedje and E. Bekell, 2000.
Comparison of multivariate methods for the analysis of genetic resources and adaptation in Phytolacca dodecandra
using RAPD. Theor. Applied Genet., 101: 1145-1154.CrossRef | Direct Link |
Sharma, R.N., 1991.
Contribution of Ayurveda towards management of bronchial asthma. Sachitra Ayurveda, 44: 349-352.Direct Link |
Souframanien, J. and T. Gopalakrishna, 2004.
A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers. Theor. Appl. Genet., 109: 1687-1693.CrossRef | Direct Link |
Tembe, R.P. and M.A. Deodhar, 2010.
Chemical and molecular fingerprinting of different cultivars of Pelargonium graveolens
(L'Herit.) viz., Reunion, Bourbon and Egyptian. Biotechnology, 9: 485-491.CrossRef | Direct Link |
Tertivanidis, K., O. Koutita, I.I. Papadopoulos, I.S. Tokatlidis, E.G. Tamoutsidis, V. Pappa-Michailidou and M. Koutsika-Sotiriou, 2008.
Genetic diversity in bean populations based on random amplified polymorphic DNA markers. Biotechnology, 7: 1-9.CrossRef | Direct Link |
Vega, K.G., M.G. Chavira, O.M. de la Vega, J. Simpson and G. Vandemark, 2001.
Analysis of genetic diversity in Agave tequilana
var. Azul using RAPD markers. Euphytica, 119: 335-341.CrossRef | Direct Link |
Verma, A., N. Kumar and S.A. Ranade, 2004.
Genetic diversity amongst landraces of a dioecious vegetatively propagated plant, betelvine (Piper betle
L.). J. Biosci., 29: 319-328.CrossRef | Direct Link |
Virk, P.S., B.V. Ford-Lloyd, M.T. Jackson and H.J. Newbury, 1995.
Use of RAPD for the study of diversity within plant germplasm collections. Heredity, 74: 170-179.CrossRef | PubMed | Direct Link |
Welsh, J. and M. McClelland, 1990.
Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res., 18: 7213-7218.PubMed | Direct Link |
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990.
DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535.CrossRef | PubMed | Direct Link |
Zietkiewicz, E., A. Rafalski and D. Labuda, 1994.
Genome fingerprinting by Simple Sequence Repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 20: 176-183.CrossRef | PubMed | Direct Link |
Sneath, P.H.A. and R.R. Sokal, 1973.
Numerical Taxonomy. 1st Edn., W.H. Freeman and Co., San Francisco, USA., ISBN-10: 0716706970, Pages: 573
Ranade, S.A., A. Kumar, M. Goswami, N. Farooqui and P.V. Sane, 1997.
Genome analysis of amaranths: Determination of inter- and intra-species variations. J. Biosci., 22: 457-464.CrossRef | Direct Link |