Leaflets 12: 476-480. 2008.
Species Relationship of Cinnamomum
Species Using RAPD Marker Analysis
Priya joy1 and M. Maridass2
for Biodiversity and Biotechnology, 2Animal Health Research Unit
St. Xavier’s College (Autonomous), Palayamkottai-627002, Tamil Nadu,
Corresponding author: E-mail
Issued 13 July 2008
Genetic inter-relationship of nine Cinnamomum species was estimated using
randomly amplified polymorphic DNA (RAPD) markers. The 15 selected
RAPD primers out of 2 primers were amplified in all cinnamon species. Cinnamomum verum has very close
similarity (87%) with C. citronella
and another two species of C. camphora
and C. glucens. Our results
suggested that genetic relationships in Cinnamomum
species using RAPD banding data may be useful for plant improvement and an
efficient way to conserve genetic resources of Cinnamomum species, in addition to their effective medicinal
Key Words: Lauraceae;
Cinnamomum; DNA; RAPD; Southern India.
This manuscript examines the
standardization of RAPD marker and genetic relationships of Cinnamomum species, a commercially
valuable source of camphor, cinnamaldehyde
and safrole oil in the world. Cinnamomum
the largest genus in the
Lauraceae, it has comprises 250 species, which are distributed in India,SriLanka and Australia. Cinnamomum was first introduced in India from Sri Lanka. Nineteen species of Cinnamomum have been found on the South India. Many species of Cinnamon yield a
volatile oil on distillation. The most important Cinnamon oils in world trade
are those from C. zeylanicum, C. cassia and C. camphora. The
other species given oils, which are utilized as sources for chemical isolates.
However, a number of other cinnamomum species are distilled on a much smaller
scale and the oils used both locally or exported (The Wealth of India,
1992).The major compounds present in both stem-bark and root bark are cinnamaldehyde (75%) and camphor (56%),
respectively (Senanayake et al., 1978). Cinnamon bark oil possesses
the aroma of the spice and a sweet and pungent taste. It is employed mainly
in the flavorings industry where it is used in meat and fast food seasonings,
sauces, pickles, baked goods, confectionery, cola-type drinks, tobacco
flavors and dental and pharmaceutical preparations. Many
species of Cinnamomum are known to contain safrole (Bin Jantan
and Goh, 1992), and concern that this beverage
contains a known carcinogen led to this study. Recently global
interest in oriental medicine, production of those plants has grown even more
over the following years. Since many species and varieties exist, development
of molecular markers would be important for quality assessment in the medicinal
industry (Sang-Bok Lee, et
During the last decade several
novel DNA-markers (RAPD, RFLP, SSR, ISSR
etc.) have been rapidly integrated into the tools available for genome
analysis. Salimath et al., (1995) has
been used for DNA fingerprinting and assessing genetic diversity. Presence or absence of DNA bands in the gel may be used as
RAPD markers to study close genetic relationship (Sang-Bok Lee, et al., 2000), inter-
and intra-specific genetic variations (M’Ribu and Hilu 1994), for the
identification of specific genes (Paran et al. 1991; Martin et al. 1991), and
to study the pattern of gene expression (Valle et al. 2000).
Materials and Methods
Young leaves of nine Cinnamomum species
viz.,C.camphora C. cassia, C. riparium,
C. macrocarpum, C. perrotteiti, C. wightii C. citronella, C. glaucense, C.
verum C.malabaratum were collected from Southern India and preserved at – 80°C.
Stored leaves were pulverized in liquid nitrogen and DNA
was extracted from each plant of Cinnamomum
species according to the method described by Doyle and Doyle (1987). Total DNA
was quantified spectrophotometrically and samples yielding good quality (A260/A280
ratio 1.7–1.9) and as well as
visually by ethidium bromide staining on 0.8% agarose gel electrophoresis.
A set of random 15
primers was purchased from a commercial source (Operon Technologies, Alameda,
Calif.). After initial tests, 15 primers (OPA-10; OPA-12; OPA-15; OPA-17;
OPB-01; OPB-15; OPC-01; OPC-15;
OPE-02; OPE-06; OPE-07; OPE-10; OPE-20; OPF-13; and OPF-20) were used for
further studies. PCR reactions were performed according to the protocol of Williams et al.
(1990). Briefly, DNA samples of the 10 Cinnamomum species of each individuals
plants were adjusted to 20 ng/µl and used in the amplification reactions with
a final volume of 25µl containing 1µl of DNA, 2 µl of primer (40
µM), 1 µl of dNTPs (10 mM), 0.2 µl Taq DNA polymerase (5 U/µl), 3
µl PCR buffer, 1.5 µl of MgCl2 (25 mM) and
16.3 µl dionized water. DNA amplification was
carried out using a eppendrof
thermocycler programmed with 3 min at 94°C for initial denaturation,
followed by 35 cycles of 54s at 94°C, 45 sec at 43°C, 2 min at 72°C, and a
final 5 min extension at 72°C. After amplification, the DNA fragments were
separated by electrophoresis for about 2 h under constant voltage (60 V) in
1.8% agarose gel submersed in 1X TBE buffer. All PCR experiments were done at least
twice and the best gels of the replicates were used for band scoring. The gels were stained with ethidium bromide solution and
observed under ultraviolet light. Each gel was photo documented using the
image capturing system bioprint. A 1 kb fragment size marker was used as a reference to
allow comparison among the different gels (Gibco 1kb ladder).
The amplified bands were scored as 1 and 0 based on
band (allele) presence and absence, respectively. Sizes of amplified bands
were estimated using Gel Pro analyzer software. The
binary data set was used to calculate the pairwise Jaccard similarity index
and to assemble the corresponding similarity matrix. The matrix obtained was
used to generate a dendrogram using the UPGMA method (Unweigthed Pair Group Method
Arithmetical Means). The distances in the dendrogram were compared with the
genetic distances between genotype pairs to calculate the cophenetic
correlation. All the analyses were performed with the aid of the 1.70 version
of the NTSYS-pc computer program.
Results and Discussion
Fifteen primers were
used in this study of RAPD marker analysis to standardization of suitable
specific primers amplifying the genetic materials of Cinnamomum species. All primers but two (OPE-20, and
OPE-13) yielded maximum amplification products with all Cinnamomum species. Primers
OPE-20 and OPE-13 amplifying
all Cinnamomum species were
produced many bands overall ranging in size 600 - 2600bp. The primers amplified DNA
products from each Cinnamomum
species generating reproducible band patterns. Primers OPE-20, and OPF- 13 generated
uncommon band with DNA from some of the
specimens, along with several common bands with each specimen .Primer OPE-20
was amplified in C.verum very close similariety (87%) with C.citronella, C.camphora
and C.glucens. Several
polymorphic bands were detected. The remaining primers gave patterns that
were identical or had differences too small to provide information on the
genetic diversity. They could hardly distinguish the nine Cinnamomum
species slightly different all Cinnamomum
species. A phylogenetic tree comprising a total of 9 Cinnamomum species RAPD marker was constructed as shown in Figure
correlation coefficient calculated between RAPD when using the similarity 87%
when using the dendrograms.
Fig. 1: Dendrogram showing OPA
- 20 primers using analysis of inter species relationship of Cinnamomum species
Cinnamomum species is a valuable spices tree in India, Sri Lanka Asia, and
Australia. Driven by commercial incentives, the wild population of this tree
has been threatened with depletion in recent years due to excessive
harvesting. The present study was preliminary attempt to develop RAPD
primers to distinguish the nine Cinnamomum
species showed that a more difficult screening of primers has to
be done before RAPD markers can be developed. Chatti et al. (2003) work done on
the genetic diversity and the phylogenetic relation between 17 ecotypes
analyzed by using the technique of RAPD marker. This study showed a
significant morphological variation and a large genetic diversity within and
among cultivars. Lately, this technique has been used to study the
genetic relations between the different species of coffee and to determine
the relationship between hybrids (Paulo, 2003). The dendrogram supports the suggestion that similar
variation of C. verum and C. citronela and also may be
synonymous with other two species of C.
riparium and C. wighti .
However the variation in terms of length 600-2600 bp and nucleotide the
frequency of insertions and deletions is too large for phylogenetic analyses.
We ould like to thank
the State Forest Department, Tamil Nadu and Kerala for permission to collect
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