Leaflets 12: 609-13. 2008.
of Inter-Species Relationships of Ocimum Species Using RAPD Markers
*Harisaranraj R., **R. Prasitha, *S. Saravana Babu and *Suresh K.
*Department of Plant Biology and Plant
Erode. (T.N.) INDIA
**Vivekananda College of Arts and Science
College for Women, Trichengode. (T.N.)��
Genetic inter-relationship of Seven Ocimum species was estimated using
Random Amplified Polymorphic DNA
(RAPD) markers. The 15 selected RAPD primers out of 2 primers were amplified
in all Ocimum
species. O. basilicum
has very close similarity (89%) with O. tenuiflorum and another two species of O. gratissimum and O. micranthum. Our results suggested
that genetic relationships in Ocimum species using RAPD banding data may be useful for
plant improvement and an efficient way to conserve genetic resources of Ocimum species,
in addition to their effective medicinal uses.
Key Words: Lamiaceae, Ocimum,
The purpose of the
present study was to use RAPD markers to evaluate the genetic relationships
between seven Ocimum
belongs to the Lamiaceae family, which has close to
252 genera and 6700 species (Mabberley, 1997) most
of which are used as medicine (Wren, 1968). The leaves are often hairy and
posses epidermal glands which secrete volatile oils giving characteristic
scents to many of the species. The essential oils found in leaves, seeds, flowers
and roots of Ocimum species are used
as medicine. Under in vitro conditions, the oils have shown to have
antibacterial activity against gram positive bacteria: Staphylococcus aureus (ATCC
25923), Bacillus species and gram negative bacteria: Escherichia
coli (ATCC 25922), Salmonella typhi, Pseudomonas aeruginosae,
Proteus mirabilis, Klebsiella pneumoniae (ATCC
27853), Salmonella enteriditis, Shigella flexineri, and
pathogenic fungus namely Candida albicans (Nakamura
et al., 1999; Matasyoh et al., 2007). Because of
its potential as a traditional medicine, incorporation of O. species into
agro forestry systems would not only make the species accessible to the
majority of the rural population that uses it but also contribute to its
genetic conservation. However, before widespread domestication of the species
is implemented, it would be important to determine its genetic diversity in India
so that useful genotypes that could be used as cultivars by farmers can be
selected thereby also facilitating the efficient conservation, management and
utilization of the species genetic diversity. There are techniques available
for assessing genetic diversity at molecular level. Those are morphology,
biochemical and more recent DNA
technology. The application of DNA technology
in agricultural research has progressed rapidly over the last twenty years,
especially in the area of cultivar identification and characterization (Nybom, 1990) as well as determination of population
diversity in many plant species (Lei et al., 2006; Chen and Yang, 2004; Nan
et al., 2003; Ipek and Madison, 2001; Muluvi et al., 1999; Cardoso et
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 al, 2000). 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 seven
viz., O. tenuiflorum, O.
americanum, O.basilicum, O. gratissimum, O. micranthum, O. kilimandscharicum ,O. campechianum �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 Ocimum 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
RAPD Marker analysis
A set of random 15 primers was purchased
from a commercial source (Sigma Aldrich, Bangalore.).
After initial tests, 15 primers (OPA-08; OPA-10; OPA-11; OPA-12; OPA-15;
OPB-03; 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 7 Ocimum species of each individuals plants were adjusted to 20 ng/�l and used in the amplification reactions with a
final volume of 25�l containing 1�l of DNA, 2 �l of primer (40 �M), 1 �l of dNTPs (10 mM), 0.2 �l Taq DNA polymerase (5 U/�l), 3 �l PCR buffer, 1.5 �l of MgCl2 (25 mM) and 16.3 �l dionized water.
DNA amplification was carried out using
Corbett Research thermocycler programmed with 3 min
at 94�C for initial denaturation, followed by 35
cycles of 54sec 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 1 hour under constant voltage (50 V) in 1.5% 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 (Genei
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 2006 version of the Non linear Dynamics -pc
Results and Discussion
Fifteen primers were used in this study of RAPD marker analysis to
standardization of suitable specific primers amplifying the genetic materials
species.� All primers but two (OPE-20,
and OPF-13) yielded maximum amplification products with all Ocimum
species.� Primers OPE-20 and OPF-13
amplifying all Ocimum
species were produced many bands overall ranging in size 800 - 2800bp. The
primers amplified DNA
products from each Ocimum
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 O. tenuiflorum very close similarity (89%) with O.basilicum, O. gratissimum and O. micranthum. 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 seven Ocimum species
slightly different all Ocimum
species. The PCR amplified band pattern of
seven species of Ocimum
species were shown in Figure.1 and the phylogenetic
tree comprising a total of 7 Ocimum species RAPD marker was constructed as shown in
Figure.2.� The correlation coefficient
calculated between RAPD when using the similarity 89% when using the dendrograms.
Fig. 1. PCR amplified band pattern of
1. O. tenuiflorum 2.O. americanum
O.basilicum 4.O. gratissimum
O. micranthum 6.O. kilimandscharicum 7. O. campechianum
Ocimum species are valued as spice plants in India.
Driven by commercial incentives, the wild population of this plant 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 seven Ocimum 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
and on nine species of Cinnamomum species by (Priya
joy et al, 2008). 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 O.basilicum and O. gratissimum and also may be synonymous with other two
species of O. tenuiflorum
and O. micranthum.
However the variation in terms of length 800-2800 bp
and nucleotide the frequency of insertions and deletions is too large for phylogenetic analyses.
- Cardoso SRS, Eloy NB, Provan J, Cardoso
MA, Ferreira PCG (2000). Genetic differentiation of Euterpe
edulis Mart populations estimated by AFLP
analysis. Mol. Ecol. 9: 1753-1760.
X, Yang J (2004). Species � diversified plant cover enhances orchard
ecosystem resistance to climatic stress and soil erosion in subtropical
hillside. J. Zhejiang. Uni. Sci. 5(10): 1191-1198.
and Doyle (1987). A rapid DNA
isolation procedure for small quantities of fresh leaf tissues. Phytoch. Bull. 19: 11-15.
- Ipek M, Madison L (2001). Genetic Diversity in
Garlic (Allium sativum
L.) as assessed by AFLPs and Isozymes. American Society for Horticultural Science
98th Annual Conference & Exhibition � 22 - 25.
Y, Gao H, Tsering T, Shi S, Zhong Y
(2006). Determination of genetic variation in Rhodiola
crenulata from the Hengduan
Mountain Region, China
using inter-simple sequence repeats. Genet. Mol. Biol. Sao Paulo (29):
- M�Ribu, H.K., and Hilu,
K.W. 1994. Detection of interspecific and intraspecific variation in Panicum
millets through random amplified polymorhic
Theor. Appl. Genet. 88: 412�416.
- Mabberley DJ (1997). The plant Book: A portable
Dictionary of the Higher plants (second edition). Useful, dictionary of
genera and families of angiosperms. In: FREMONTIA. J. California Native
Plant Soc. 30(2): 1-36, 2002.
- Matasyoh LG, Matasyoh JC, Wachira FN, Kinyua MG, Muigai Thairu AW, Mukiama TK (2007). Chemical composition and
antimicrobial activity of the essential oil of Ocimum
gratissimum L. growing in Eastern
Kenya. Afr. J. Biotechnol. 6(6): 760-765.
- Muluvi GM, Sprent JI, Soranzo N, Provan J, Odee D, Folkard G, McNicol JW, Provan J,
Powell W (1999). Amplified fragment length polymorphism (AFLP) analysis
of genetic variation in Moringa. Mol. Ecol. 8:
- Nakamura CV, Nakamura TV,
Bando E, Melo AFN, Cortez DAG, Dias Filho BP (1999). Antibacterial
activity of Ocimum gratissimum L. essential oil. Mem. Inst. Oswaldo Cruz.
P, Shi SH, Peng ST ,Tian Cj ,Zhong Y (2003). Genetic diversity in Primula obconica
(Primulaceae) from central and south-west China
as revealed by ISSR markers. Ann. Bot. 91:
- Nybom H (1990). Application of DNA
fingerprinting in plant breeding. Proc. Intern. DNA
Fingerprinting Symposium. Bern.
- Paran, I., Kesseli, R., and Michelmore,
R. 1991. Identification of RFLP and RAPD markers linked to downey
mildew resistance gene in lettuce using near-isogenic
lines. Genome, 34: 1021�1027.
- Priya joy and M. Maridass. Ethnobotanical
Leaflets 12: 476-480. 2008.
- Salimah, S.S., De Oliveira,
A.C., Godwin I. D (1995). Assessment of genomic origin and
genetic diversity in the genus Eleusine with DNA
markers. Genomic. 38: 757�763.
- Sang-Bok Lee, and Soren K. Rasmussen, 2000. Molecular markers in some medicinal
plants of the Apiaceae family. Euphytica, 114: 87�91.
Wealth of India,
(1992). A Dictionary of Indian Raw materials and Industrial products, III
Publications and Information Directorate, New Delhi, p. 582-590.
- Valle, P.R., Souza, M.B.,
Pires, E.M., Silva, E.F., and Gomes, M.A. 2000. Arbitrarily
fingerprinting of RNA and DNA
in Entamoeba histolytica.
Rev. Inst. Med. Trop. Sao Paulo, 42(5):
JGK, Kubelik AR, Livak
KJ, Rafalski JA and Tingey
SV (1990) DNA
polymorphisms amplified by arbitrary primers are useful as genetic
markers. Nucleic Acids Research 18:6531-6535.
RW (1968). Potter�s New Cyclopedia for Botanical Drugs and Preparations
by Wren RC. Re-edit by Wren RW. Potter and Clarke, Ltd. Health Science