Journal of Academia and Industrial Research (JAIR)

Volume 2, Issue 2 July 2013

83
ISSN: 2278-5213

RESEARCH MANUSCRIPT

Genetic variation between molly fishes Poecilia latipinna and

Poecilia sphenops using RAPD assay
1*

1,2

M. Shanmughavalli , K. Narmathanathiya , C. Arulvasu and D. Chandhirasekar

Dept. of Advance Zoology and Biotechnology, Quaid-E-Millath Govt. College for Women, Chennai-600002
3,4
Dept. of Zoology, University of Madras, Guindy Campus, Chennai-600025
shanmughavalli@yahoo.com*; +91 9444781605

______________________________________________________________________________________________

Abstract
Investigation of genetic variations for Poecilia latipinna and Poecilia sphenops from different locations of
Chennai, Tamil Nadu was performed using Random Amplified Polymorphic DNA (RAPD) assay. High degree
of polymorphism was observed, suggesting the degree of genetic variability between P. latipinna and
P. sphenops. The low levels of genetic variation within-species are due to their limited migration and pair
fidelity mode of reproduction. Random primer RAPD3 seems to be a good candidate for developing markers.
The dendrogram obtained from RAPD clearly depicts that Poecilia sp. are closely related to each other
where P. latipinna shows 57% of variation from dendrogram scale.
Keywords: Genetic variations, Poecilia sp., RAPD, polymorphism, random primer, dendrogram, marker.

Introduction
In recent years, a wide range of new molecular
techniques have been explored and reported for fishes
and shellfishes (Lehmann et al., 2000; Jayasankar,
2004). Several DNA techniques for evaluating genetic
variability in fish species are available and are widely
used (Harris et al., 1991; Mjolnerod et al., 1997;
Coughlan et al., 1998; Wasko and Galetti, 2002; Barman
et al., 2003; Matoso et al., 2004; Jayasankar, 2004).
One such technique is the Random amplified
polymorphic DNA (RAPD) which was first introduced by
Williams et al. (1990). It is a technique based on the PCR
amplification of discrete regions of genome with short
oligonucleotide primers of arbitrary sequence (Welsh and
McClelland, 1990; Williams et al., 1990). It utilizes single,
arbitrary, decamer DNA oligonucleotide primers to
amplify regions of genome based on the polymerase
chain reaction (Hadrys et al., 1992; Williams et al., 1993).
The characters assessed through RAPD are useful for
genetic studies because they provide various types of
data-taxonomic population, inheritance pattern of various
organisms including fishes (Brown and Epifanio, 2003;
Degani, 2004; David and Pandian, 2006).
Genetic variability on an endangered Neotropical fish
species were determined by RAPD analysis which
reveals variation between the species (Wasko and
Galetti, 2002). Genetic monitoring of the Amazonian fish
matrincha Brycon cephalus were studied using RAPD
markers which determine the usefulness of genetic
management and biodiversity conservation of this
species (Wasko et al., 2004). Genetic diversity of three
cultured populations of goldfish were studied which
revealed poor genetic diversity of goldfish (Xi-dong et al.,
2007).
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RAPD analysis of two different populations of cultured

Korean catfish Silurus asotus were determined which
revealed genetic similarity in cultured catfish populations
(Yoon and Kim, 2001). Population structure and
phylogenetic relationships among the Spanish Barbus
species were analyzed RAPD technique (Callejas and
Ochando, 2002). The patterns of morphometric and
genetic variation of three species of Garra were studied
using RAPD analysis which revealed that G. mullya and
G. kalakadensis hold similar characters compared to the
other congener, G. gotylastenorhynchus (Dhinakaran
et al., 2011). Genetic variations among Nile Tilapiine
fishes were assessed by RAPD revealed that the
different primers have different performances for
evaluation of genetic polymorphism (El-Alfy et al., 2009).
DNA fingerprinting of eight cyprinid fish species of Iraqi
Inland waters using RAPD-PCR revealed that the eight
cyprinid taxa are distinctive species (Faddagh et al.,
2012). Inheritance of RAPD markers in the Guppy fish,
Piecilia reticulata was assessed using RAPD analysis
which determined higher genetic variability between the
two varieties and low intra variety genetic variability (Foo
et al., 1995). Ten freshwater fishes Mylopharyngodon
piceus, Ctenopharyngodon idellus, Hypophthalmichthy
molitrix, Aristichthys nobilis, Cyprinus carpio, Carassius
auratus, Mygalobrama amblycephala, Silurus asotus,
Myxocyprinus asiaticus and Peteobargrus fulvidraco
were identified using RAPD analysis (Huai et al., 1998).
Morphological and genetic variability of Malaysian
Channa sp. were assessed using RAPD technique
(Husin and Norainy, 2007). The genetic structure of the
fish Astyanax altiparanae populations were analyzed
using RAPD technique which revealed structures
regarding fish handling and conservation programs
(Leuzzi et al., 2004).

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84

Gene mapping and genetic variation of channel catfish

(Ictalurus punctatus) and blue catfish (Ictalurus furcatus)
were determined using RAPD analysis which shows high
level of polymorphism (Liu et al., 1999). Genetic diversity
of three ornamental reef fishes from the Brazilian coast
were determined by RAPD technique revealed genetic
variation in the three species were related to
intra-population diversity (Affonso and Galetti, 2007).
Genetic variation of three Norwegian Gyrodactylus
salariis populations were determined using RAPD
analysis revealed polymorphism between different
populations of G. salariis (Cunningham and Mo, 1997).
From the above literature, the reports on the genetic
variability are very scanty in molly fishes. Hence, the
present study is aimed to analyze genetic variability in
Poecilia latipinna and Poecilia sphenops species using
RAPD analysis.

Materials and methods

Collection and maintenance of fishes: Poecilia latipinna
and P. sphenops were collected at two different
geographical locations from Kolathur (13071.25N,
8001241.18E) and Perugalathur (1205418.36N,
800541.28E), Chennai. They were brought to the
laboratory in a plastic bag containing fresh water with
continuous aeration and maintained in glass tank
(20 x 10 cm) with 15 cm column of fresh water.
The water was aerated continuously and changed every
day. These fishes were fed with fish feed and were
acclimatized for 2-3 d in prevailing room temperature.
Only healthy fishes weighing 1.82 g were used for
experimental analysis.
Quantitative and qualitative determination of DNA:
Approximately 0.3 g of molly fish muscle tissues were
dissected and homogenized with 2 mL of lysis buffer and
0.01% of proteinase K using mortar and pestle. The DNA
was isolated according procedure of Wu et al. (1995).
The DNA pellet was washed with 70% ethanol and
suspended in 20 L of 1xTE buffer and stored at -20C
until required. DNA quality was assessed by using
Double beam Spectrophotometer R2203 according to
Sambrook et al. (1989) at the absorbance ratio of 260
and 280 nm providing a value of 1.8 which determines
pure DNA preparation.
The concentration of DNA in the sample was calculated
using the given formula:

Ribonuclease (RNase) treatment: DNA samples (20 L)

were made up to 400 L with 1xTE buffer (pH 7) and
20 g/mL concentration of RNase solution were added to
the reaction tubes and the samples were incubated at
37C for 1 h. The supernatant were extracted once with
an equal volume of phenol: chloroform: isoamyl alcohol
(25:24:1) and centrifuged (12000 rpm, 10 min).
The samples were precipitated with double the volume of
absolute alcohol (12000 rpm, 15 min). The DNA pellets
were air-dried and dissolved in 20 L of 1xTE buffer and
stored at -20C until required. The concentration of
extracted DNA was determined spectrophotometrically at
260 and 280 nm and electrophoretically analyzed
through 0.8% agarose gel containing ethidium bromide
stain and visualized using a UV transilluminator
(Sambrook et al., 1989).
Screening of RAPD primers: Under optimized PCR
conditions, random primers RAPD1, RAPD3, RAPD5,
RAPD6, RAPD7, RAPD8, RAPD9 and RAPD10 were
used for screening. Primers were chosen for further
studies based on the criterion that the DNA patterns
were consistent in all trials.
PCR amplification: RAPD-PCR was performed in an
Eppendorf Master Cycler personnel thermo cycler at
94C for 3 min, 94C for 45 sec, 37C for 1 min, 72C for
1 min, 94C for 45 sec (repeated 39 times), 72C for
7 min and hold at 4C. Optimal program parameters
were selected on the basis of consistent amplification at
all DNA concentrations in addition to increased intensity
and clarity of the banding pattern. The resulting products
were electrophoretically analyzed through 1.5% agarose
gels stained with ethidium bromide and visualized using
a UV transilluminator.
RAPD dendrogram: Dendrogram for RAPD was
constructed using SPSS (version 16, 2007) software tool.

Results
Quantitative determination of DNA: Spectrophotometric
analysis of the DNA samples showed the ratio of DNA
samples of P. latipinna and P. sphenops of Kolathur and
Perungalathur as 1.7032, 1.7088, 1.7078 and 1.6910
respectively. RNase treatment was done to remove the
RNA present along with genomic DNA band. Before
RNase treatment there is presence of RNA along with
genomic DNA band (Fig. 1) and after RNase treatment
there is absence of RNA band and the presence of only
the genomic DNA band (Fig. 2) were noted.

Concentration of DNA = A260 50 g dilution factor

Purity of the DNA = A260: A280 ratio = A260 /A280
DNA fragment was electrophoretically analyzed through
0.8% agarose gel containing ethidium bromide stain at
50 V and visualized using a LARK UV transilluminator
(Sambrook et al., 1989).
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Screening of RAPD primers: Under optimized PCR

conditions, random primers RAPD1, RAPD3, RAPD6,
RAPD7, RAPD8, RAPD9 and RAPD10 were used for
screening the DNA of P. sphenops (Kolathur, Chennai)
and P. latipinna (Kolathur, Chennai). Among the primers
tested RAPD1, RAPD3, RAPD7 were found to be
suitable for further investigation (Fig. 3).

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Journal of Academia and Industrial Research (JAIR)

Volume 2, Issue 2 July 2013

85

Fig. 3. Primer screening for P. latipinna and

P. sphenops DNA sample.

Fig.1. Genomic DNA samples before RNase treatment.

Kb
10000

1000

E
A

A: DNA Marker; B: P. sphenops (kolathur); C: P. latipinna (kolathur);

D: P. sphenops (Perungalathur); E: P. latipinna (Perungalathur).

Fig. 2. Genomic DNA samples after RNase treatment.

10000

RAPD analysis: A total of 65 scorable bands ranging

from 100 to 2500 bp were observed from the RAPD
analysis of P. latipinna and P. sphenops using three
oligonucleotide primers RAPD1, RAPD3, and RAPD7
(Fig. 4). The number of amplified bands in all
investigated samples was 13 bands for primers RAPD1,
13 bands for RAPD3 and 9 bands for RAPD7. Thirteen
polymorphic bands were seen in RAPD1, monomorphic
bands are absent; 11 polymorphic band and
2 monomorphic bands were seen in RAPD3;
1 monomorphic band and 8 polymorphic bands were
seen in RAPD7. The percentage of polymorphic band of
RAPD1, RAPD3 and RAPD7 is given in Table 1.

1000

Primers were chosen for further studies based on the

criterion that the DNA patterns were consistent in each
trials.

Kb

A: RAPD3, 3 Prominent bands of P. sphenops-Kolathur;

B: RAPD7, 4 Prominent bands of P. latipinna-Kolathur;
C: RAPD1, 2 prominent bands of P. latipinna-Kolathur.

A: DNA Marker; B: P. sphenops (kolathur); C: P. latipinna (kolathur);

D: P. sphenops (Perungalathur); E: P. latipinna (Perungalathur).

Fig. 4. RAPD PCR for primers RAPD1, RAPD3 and RAPD7.

A: RAPD1 PCR for P. sphenops (Kolathur); B: RAPD1 PCR for P. latipinna (Kolathur); C: RAPD1 PCR for P. sphenops (Perungalathur); D: RAPD1 PCR
for P. latipinna (Perungalathur); E: RAPD3 PCR for P. sphenops (Kolathur); F: RAPD3 PCR for P. latipinna (Kolathur); G: RAPD3 PCR for P. sphenops
(Perungalathur); H: RAPD3 PCR for P. latipinna (Perungalathur); I: RAPD7 PCR for P. sphenops (Kolathur); J: RAPD7 PCR for P. latipinna (Kolathur);
K: RAPD7 PCR for P. sphenops (Perungalathur); L: RAPD7 PCR for P. latipinna (Perungalathur); M: RAPD Marker.

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Table 1. Sequence of oligonucleotide primers, sizes and

numbers of scorable RAPD bands and percentage of
polymorphic bands resulting from RAPD analysis using
primers RAPD1, RAPD3, and RAPD7.
Percentage
Size range
of
Primer
(no. of
polymorphic
Primer
Sequence
Scorable
band (no. of
bands)
polymorphic
bands)
RAPD1 TCTCAGCATG 200-2500 (13)
100 (13)
RAPD3 TAGGTCTCTG 100-2500 (13)
84.61 (11)
RAPD7
CGGATATCG
200-1200 (9)
88.88 (8)

Fig. 5. RAPD dendrogram.

A

Table 2. Molecular diagnostic key of P. latipinna and

P. sphenops based on RAPD analysis.
RAPD
Primer
marker
Species/Location
size
300 bp
P. sphenops (Perungalathur)
RAPD1
550 bp
P. latipinna (Perungalathur)
1200 bp
P. latipinna (Perungalathur)
800 bp
P. sphenops (Kolathur)
550 bp
P. latipinna (Perungalathur)
RAPD3
1200 bp
P. latipinna (Perungalathur)
P. latipinna (Kolathur and
2500 bp
Perungalathur)
P. latipinna (Kolathur and
550 bp
Perungalathur)
RAPD7
1200 bp
P. latipinna (Perungalathur)

0.39

0.55

0.63

0.71

Coefficient

Discussion

Species specific markers: Several RAPD fragments

showed fixed frequencies in each of particular species.
Different bands were seen in P. latipinna and
P. sphenops of Perungalathur and Kolathur using primer
RAPD1, RAPD3 and RAPD7 which is represented in
Table 2.
Similarity index, genetic distance and phylogenetic
relationships: Similarity index within the same species of
molly fish was between 0.571 and 0.666 (Table 3).
The genetic distances between the molly fishes within
the same species was much lower between different
species (Table 3).
RAPD dendrogram: The phylogenetic tree constructed
from genetic distances showed that the dendrogram is
divided into two major clusters for P. sphenops and
P. latipinna molly fish species. Poecilia sphenops of
Kolathur and Perungalathur showed 71% relationship
whereas P. latipinna of Kolathur and Perungalathur
showed 57% relationship. Poecilia sphenops from
Kolathur and Perungalathur forms single cluster and
P. latipinna from Kolathur and Perungalathur forms
another
cluster.
Genetic
difference
between
P. latipinnna, P. sphenops from Kolathur and
Perungalathur is 39% based on the distance scale
(Fig. 5).

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0.47

The present study documents the genetic variability

within and between the species of molly fishes
P. latipinna and P. sphenops from Kolathur and
Perungalathur Chennai using RAPD technique.
In capture fishery, excessive exploitation combined with
poor fishery management results in the depletion of the
fishery stocks, such depletions can result in the loss of
total gene pool (Nelson and Soule, 1987; Smith et al.,
1991). In this investigation, most of the genetic
characteristics of fishes were similar and often
over-lapped with population. These data are not enough
to support the established genetic structure of the
population that often leads to taxonomic uncertainty
(Daniel, 1997; Ponniah and Gopalakrishnan, 2000).
Allozymes and mophometric analyses were used to
discriminate Hilsa populations which were collected from
nine different sites within Bangladesh (Salini et al.,
2004). RAPD was used in the preliminary inferences
concerning genetic variability between two species of
crayfish (Cambarus maculatus and Astacus astacus) and
two species of fish (Carassius auratus and Poecilia
latipinna). This technique is based on the detection of
polymorphisms, bands observed in some individuals but
absent in others, which are amplified by random primers
during the PCR. The primers RAPD1, RAPD3 and
RAPD7 were used in the present study. All the three
primers examined produced different RAPD fragment
patterns. The number of amplified bands detected varied,
depending on the primers and species. To ensure that
the amplified DNA bands originated from genomic DNA,
not from primer artifacts, negative control was carried out
for each primer species combination. No amplification
was detected in the control reactions. All amplification
products were found to be reproducible when reactions
were repeated using the same reaction conditions.
A high degree of polymorphism was observed,
suggesting a high degree of genetic variability between
P. latipinna and P. sphenops.

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87

Table 3. Similarity index and genetic distance within the species of P. latipinna and P. sphenops
analyzed by RAPD1, RAPD3 and RAPD7.
Total no. of
Percentage of
Similarity index
Genetic distance
Average no. of
monomorphic/
Species
polymorphic
within the
within the
bands per primer
Polymorphic
bands
species
species
bands
P. sphenops
6.66
1010
50
0.666
0.334
P. latipinna
8.33
1015
60
0.571
0.429
The presence of variability among populations as well
as individuals within a population is essential for their
ability to survive and successfully respond to
environmental changes (Ryman et al., 1995). Intrapopulation genetic variation in tilapia was studied using
different RAPD primers (Bardakci and Skibinski, 1994).
This technique is more sensitive than the mt-DNA
analysis, which failed to reveal variations within the
tilapia populations (Capili, 1990; Seyoum and Kornfield,
1992). Genetic variation was studied between four
different populations of Hilsa Sha from Ganga, Yamuna,
Hoogly and Narmada rivers of India using RAPD
technique (Brahmane et al., 2006). Thus, RAPD has
been used in population studies in fisheries and can be
used efficiently for variation analysis of populations with
differential degrees of geographic isolation.
The present study is the first report on the use of RAPD
markers for studying genetic variation in molly fishes.
The low levels of within-species genetic variation
exhibited in P. latipinna and P. sphenops are due to their
limited migration and pair fidelity mode of reproduction.
Similar observations were reported by Barman et al.
(2003) in carp species. The main objective of the study
was to evaluate the level of variation and to identify
species diagnostic markers of P. latipinna and
P. sphenops. RAPD as a rapid method for developing
genetic variability developed unique molecular markers
for P. latipinna and P. sphenops. Species-specific RAPD
markers were observed using three random primers.
Random primer RAPD3 seems to be a good candidate
for developing markers. The genetic distance is more
between genus than between species and the
hypothesis is also proved in this study by this marker.
This was also proved in earlier studies in Indian major
carps (Barman et al., 2003). Dendrogram was
constructed from similarity matrix values using UPGMA
algorithm. A statistical software package SPSS version
16 was used to developed dendrogram. The dendrogram
obtained from the RAPD data clearly depicts the
relationships among P. latipinna and P. sphenops.
The dendrogram divides into two major clusters
containing P. latipinna and P. sphenops together.
Poecilia sphenops sp. was closely related to each other.
Poecilia latipinna species shows 57% of variation from
dendrogram scale. This again reflects the RAPD results.

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SPSS has been used to interpret RAPD results for

various organisms like genetic diversity of cultivated
olives (Sesli et al., 2010). The SPSS as a system of
integrated packages can deepen the understanding of
the studied topic and give better insight in to solving
complex problems.

Conclusion
RAPD analysis is a rapid and convenient technique for
estimating genetic variation between P. latipinna and
P. sphenops and to generate useful genetic markers in
molly fishes. RAPD fragments observed in the two
individuals, showed a reasonable degree of genetic
variation within and between the species. The population
specific bands could not be discerned from the fragment
patterns generated. This observation clearly indicated
that, both the populations genetic similarity index and
genetic distance within the species.

Acknowledgements
Authors are thankful to UGC-SAP, New Delhi, India for
financial assistance.

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