Research Department of Botany, Pasumpon Thiru Muthuramalinga Thevar Memorial College, Kamuthi-623 604,
*Corresponding author: email@example.com
The Molecular markers have been widely used in analysing genetic diversity of species for the conservation approach. Eugenia
habitat. There were 20 Random Amplified Polymorphic DNA (RAPD) markers applied to find out genetic diversity among the
five existing populations. The Result showed that the genetic diversity (H) ranged from 0.13 to 0.28, the genetic diversity overall
) on an average was 0.39, genetic diversity within populations (Hs) was 0.31, genetic differentiation (G
populations overall loci was 0.26. Inter population gene flow (Nm) was 4.89. Highest percentage of polymorphism showed only
37.5%. This low percentage of polymorphism within the populations is attributed to small population size and reduced gene flow.
The result showed on evidence of the species become extinct due to loss of genetic richness among the population.
Genetic diversity, Eugenia singampattiana Bedd., RAPD, Critically endangered.
The Western Ghats of India is one of the ecologically
sensitive zones in the World. It represents about 4000
species of flowering plants and out of this, nearly 38
percent are endemic (Nair and Daniel, 1986). This
high level of diversity and endemism has conferred as
the hot spots status (Johnsingh, 2001). Globally, there
are 229 plant species are represented as threatened in
the Western Ghats. Among these, 39 are Critically
Endangered, 111 are Endangered, and 79 are
Vulnerable. Eugenia singampattiana is one of the
critically endangered endemic species based on
population size reduction of ≥90% over the last 10
years or three generations due to loss of habitat. It
belongs to the family Myrtaceae. It is a small
evergreen medicinal tree found at the tail end of
Southern Western Ghats regions of Tamil Nadu. The
plant has potential activity like anticancerous,
antitumerous, antioxidative, antimicrobial, antifungal,
anti inflammatory, antihyperlipidaemic and
antidiabetic agents (Viswanath
diversity for development of modern conservation
system. Intra species variation is prerequisite for
adaptive change for long term conservation of the
The Molecular markers have been widely used in
analysing genetic diversity and to find out highly
adaptive population (Warude et al., 2003). Polymerase
Chain Reaction (PCR)-based methods such as RAPD
marker system has been used because of its wide
usage of genetic diversity study and it reflects the
variation of the whole genomic DNA. It would be a
better parameter to measure the pattern of genetic
diversity of the rare and endangered plants (Lal et al.,
2011). Hence, the present study deals with the analysis
of genetic diversity of Eugenia singampattiana to find
out highly adaptive population for future conservation
The plant materials were collected from five existing
populations such as Inchikuzhi (ES
), Oothu (ES
), Karaiyar (ES
) and Kothaiyar
) . The plant was identified and checked with the
Garden and Research Institute (JNTBGRI) and the
deposited in JNTBGRI.
The total genomic DNA was extracted using the
modified CTAB method (Doyle and Doyle, 1987)
from tender uninfected leaf samples and purified
according to the Sambrook and Russel (2000).
Concentration of the purified genomic DNA in each
case was adjusted to 10 ng/ µl in different aliquots and
stored at - 4° C for PCR amplification.
1990). PCR reactions were carried out in a final
volume of 25 µl, which contained 2.5 µl 10X taq
polymerase buffer, 2.0 µl of deoxyribonucletides
(dNTPs), 3.5 µl MgCl
, 0.1 µl of taq DNA
µl of template DNA and 12.9 µl of sterile dis.H
mastercycler nexus gradient). Amplification process
contain, initial denaturation of DNA at 95ºC for 5
minutes, denaturation at 94ºC for 30 seconds,
annealing at 35ºC for 1 minute and extension at 72ºC
for 2 minutes followed by thirty five cycles and final
extension at 72ºC for 5 minutes. The amplified
products are stored at 4ºC till electrophoresis. The
PCR products were resolved by electrophoresis on 1.5
% agarose gel containing ethidium bromide along with
1 Kb ladder DNA as a standard molecular weight size
marker. The gels were visualized under UV
Amplification profiles of populations were compared
with each other. Genetic similarity matrix among
populations of each samples were calculated using the
standard coefficient method (9). The dendrogram was
constructed using the UPGMA (Unweighed Pair
Group Method with Arithmetic Average) (Sneath and
Sokal, 1973) algorithm in SHAN clustering module of
NTSYS-pc software version 1.5 (Rohlf, 1989 ). The
genetic diversity within and between populations
according to Nei’s formula (Nei, 1973) was calculated
using POPGENE package version 1.31software (Yeh,
A total of 30 arbitrary 10-mer primers screened, of
which 20 primers produced reproducible, multiple
band profiles with a number of amplified DNA
fragments that varied from 4 to 10. A sum of 152
polymorphic bands was observed. The size of the
RAPD fragments varied from 0.2 to 1.0 kbp (Plate 2).
Plate 2. RAPD-PCR fingerprinting of Eugenia singampattiana
Genetic and gene diversity measures were calculated
according to Nei’s index using POPGENE software
and results were depicted in the Table1. The mean
genetic heterozygosity or diversity (H) ranged from
0.1320 to 0.2863. The ES
population was found to be
population displayed the
population revealed intermediate diversity (0.14). The
to 1.6250. The ES
population was found to be least
population displayed the
The mean effective number of alleles (Ne) was 1.3103.
The highest was 1.5433 in ES
population and the
and the average was 0.2431. The mean number of
polymorphic loci (NPL) was 53 and ranged from 49 to
57 for all the accessions. The highest percentage of
polymorphism was 37.50 in ES
– Inchikuzhi; ES
– Kannikatti; ES
– Oothu; ES
– Karaiyar; ES
The overall observed and effective number of alleles
was about 1.500 and 1.3365 respectively and the
overall percentage of polymorphic loci was 50. Nei’s
overall genetic diversity or heterozygosity was 0.1958.
The genetic distance between the population ranged
from 0.2117 to 0.4389 and the genetic identity ranged
from 0.6447 to 0.8092 (Table 2). The average gene
diversity within populations (Hs) was 0.31, the highest
Hs was 0.35826 and the lowest Hs was 0.1453. The
total diversity (H
) ranged from 0.2179 to 0.4835 and
the average was 0.3924. The mean genetic
) between populations over all loci
was 0.26 and the G
ranged from 0.1532 to 0.4620.
The gene frequency ranged from 0.7394 to 0.7527 and
the average was 0.7464. The average gene flow from
one population to other population (Nm) was 4.8989
while the lowest was 0.6677 and the highest were
21.88 (Table 3).
Table 2. Nei’s unbiased measures of Genetic distance and Genetic identity of E. singampattiana
Nei's genetic identity (above diagonal) and genetic distance (below diagonal)
In order to study the correlation between populations,
UPGMA algorithm was used to draw a dendrogram
for the five populations of E. singampattiana (Fig 2).
A Jaccard’s matrix was used to produce a dendrogram
based on SI, which showed distinct separation of the
collected accessions from five locations into two
major groups having 77 % similarity. Among the two
major clusters, the accession belonging to the lower
cluster (LC) was collected from ES
, while accessions
. Further the accessions of
the UC was grouped into two major sub clusters
(USC1 &USC2) having 79 % similarity. The upper
sub cluster 1 (USC 1) was sub divided into two sub
clusters USC1A which was collected from ES
having 83% similarity.
collected from ES
and USC2B collected from ES
and they showed 93 % similarity.
The polymorphism percentage are low due to small
because geographically restricted
species showing lower levels of genetic variation than
widely distributed species (Hamrick and Godt 1996,).
on only lat. l8°33’N to 8°42’46’’N and between long.
77°17’55’’E to77°21’37’’E with two fragmented
population (Gobalan and Henry, 2000).
was also reported (Catana et al., 2013, Bantawa et al.,
2011) in endangered plants. The habitat type is another
important factor shaping the degree of genetic
diversity and differentiation (Shikano et al., 2010).
Because of the poor knowledge of the historical
distribution, it is difficult to explain the historical
factors that may have contributed to the low genetic
variation in this species. This species grows on sandy
different altitudes from 300 m to 900 m (Sarcar et al.,
2006). The average gene flow from one population to
other population (Nm) was 4.8989 and gene flow can
occur through seed dispersal (Ellstrand, 1992). In field
observation, number of seedlings were very less
because seed germination requires 65 to 85 days
1999). The low percentage of
polymorphism, reduced gene flow due to small
population size and loss of genetic richness among the
population are molecular evident of this species
Analysis of RAPD data can be used to detect genetic
variation of E. singampattiana. It showed that the
exhibited low percentage of
polymorphism, small population size, reduced gene
flow and other historical factors disclose the alarm of
species extinction. Among the natural populations, the
) accession has possessed the highest
can be adapted for the long term conservation through
The authors are thankful to University Grant
Commission (UGC), Government of India, New Delhi
for financial assistance under Major Research Project.
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