Genetic Diversity Between Two Populations of Heteropneustes fossilis (Bloch) Using RAPD Profile
The present study was aimed to elucidate its genetic diversity based on RAPD markers from 6 individuals belonging to two ecological habitats i.e., upper lake and lower lake of Bhopal. Ten random 10-mer primers were scored in each of the individuals from two locations out of which three primers, which gave polymorphism, were selected for Polymerase Chain Reaction (PCR) and used in the final RAPD analysis. The complementary approach of RAPD was used to evaluate the genetic diversity among all the accessions using 3 highly polymorphic primers. Using these primers, 128 scorable DNA fragments were found, of which 24 (18.75%) were polymorphic. By comparing RAPD banding patterns, small variations were found between and within the populations. The present study yielded data elucidating the usefulness of complementary approaches to make diversity analysis more explanatory and purposeful for optimum genetic amelioration and effective conservation of its genotypic variability.
The freshwater resources of India are currently experiencing an alarming decline
in fish biodiversity due to several factors and a result, a sizeable portion
of freshwater fishes have been categorized as threatened. This emphasizes an
immediate need for initiating research and actions for alternative management
techniques to protect these aquatic systems. Several species of fish have declined
and some have become endangered. Therefore, in order to prioritize fish species
for taking conservation action, there is an urgent need to assess the present
status of freshwater fish of the Central India. Out of about 700 freshwater
fishes reported from Indian inland waters, 320 were assessed in a workshop in
1998 jointly organized by National Bureau of Fish Genetic Resources, Lucknow
and Zoo Outreach Organization, Coimbatore, India (CAMP, 1998).
Of these 320 fish species assessed, about 166 were from Central Region and among
these 21 were categorized as endangered, 01 critically endangered, 27 vulnerable,
50 at lower risk or near threatened, 8 at lower risk of least concern and 59
species were not evaluated (CAMP, 1998). The base list
prepared by National Bureau of Fish Genetic Resources was assessed in a workshop
held in 2006 for Central India, where 138 species were from Madhya Pradesh (CAFF,
2006). The overall assessment of this workshop indicates that 01 species
were under critically Endangered Category (CE), 14 species under Endangered
Category (EN), 26 under vulnerable (VU), 53 under Data Deficient (DD), 37 under
lower risk of near threatened (LR-nt) and 08 under lower risk of least concern
(LR-lc) category. Total of 41 (56.58%), 29 (34.8%) and 18 (11.16%) species were
categorized as threatened in Madhya Pradesh, Chattishgarh and Rajasthan, respectively
(CAFF, 2006). This observation clearly indicates that,
fishes are declining at a faster rate in Madhya Pradesh than the other two states
of Central India.
Genetic markers are important instruments for the study of fish populations
and fish lots. The development of molecular methods has permitted genomic analysis
and made it possible to analyze existing variations, both in the regions which
encode genes, as well as regions with unknown functions (Jayme
et al., 2008). Genetic monitoring is ideal for use in a reproduction
program with the aim of genetic conservation (i.e., stocking). Molecular markers
are realistic and useful tools for the investigation and monitoring of genetic
conditions both in native populations and in captive lots (Alam
and Islam, 2005). The RAPD (Random Amplified Polymorphic DNA) and microsatellite
markers are among the molecular markers used to analyze genetic diversity of
fish. Both of these markers may be analyzed by PCR (Polymerase Chain Reaction).
Catfish is a commercially important warm water fish species, which is distributed
all over the world. Consequent to the rapid increase in hatchery-reared catfish
production, there is a need to understand the genetic composition of natural
catfish populations in order to evaluate the range and extent of latent genetic
effects induced by hatchery operations. Little information is available on the
phylogenetic relationship among the populations of catfish of India. Rapid amplified
polymorphism NDA technique is the one of the most frequently used molecular
methods for taxonomic and systematic analyses of various organisms (Bartish
et al., 2000) and has provided important applications in catfish.
In the present study germplasm collection of Heteropneustes fossilis
(Bloch), a catfish freshwater fish with edible fish, was carried out from two
locations of Bhopal i.e., upper lake and lower lake and find out intra-specific
MATERIALS AND METHODS
Fish Sampling Sites and Morphometric Measurements of Fishes
The present study was carried out from July, 2008 to December, 2008. A total
of 06 specimens of Heteropneustes fossilis were collected from both the
locations (upper lake and lower lake, Bhopal) with the help of castnet by local
fishermen for morphometric measurements and estimated genetic variations. All
the fish specimens were kept in the iceboxes and brought to the MPCST laboratory
for further study. For the morphometric measurements, total 14 parameters i.e.,
total length, standard length, body weight, body width, body depth/height, head
length excluding snout, width of head, snout length, eye diameter, length of
caudal peduncle, length of anal fin, length of pelvic fin, length of pectoral
fin, height of caudal peduncle were taken. Fish specimens were morphologically
identified with taxonomic keys (Shrivastava, 2000; Jayaram,
1999). The muscle, liver and brain tissues were isolated from freshly caught
fishes and preserved at -20°C for further use.
For the isolation of total genomic DNA, a short procedure was applied according
to a modified protocol of Wu et al. (1995).
Tissues (100 mg) were placed in a 1.5 mL micro-centrifuge tube (Eppendorf, A.G.,
Humberg, Germany) and homogenized by using Eppendorf micro-pestle. In the homogenized
tissue, 0.5 mL of lysis buffer (4 mM NaCl, 0.5 mM EDTA, 0.1% SDS and 0.02 NP
40) and 0.01% proteinase-K were added, gently mixed and incubated at 55°C
on dry bath (Genei, model-SLM-DB-120) for 45-60 min for complete lysis of cells.
After incubation, 500 μL phenol: chloroform: isoamyl alcohol (25:24:1)
was added, mixed gently and centrifuged at 10,000 rpm at room temperature (High
Speed Brushless Centrifuge, MPW-350R, Poland) for 5 min. The supernatant was
then transferred to a new micro-centrifuge tube and half volume of 7.5 M ammonium
acetate and two volume of 100% chilled ethanol was added, mixed well and centrifuged
at 10,000 rpm for 5 min at room temperature. One microliter of 70% ethanol was
added to the tube for washing. Again centrifuged the sample for 10 min at 10,000
rpm at 4°C. The DNA pellet was then washed with 70% ethanol, dried and dissolved
in a Tris-EDTA buffer (10 mM Tris Hcl, 1mM EDTA, pH 7.6). UV-VIS Spectrophotometer
(Nano-Drop ND-1000, USA) was used to check quality as well as quantity of isolated
DNA. The concentration of extracted DNA was adjusted to 50 ng μL-1
for PCR amplification.
In this study, 10 commercially available RAPD primers of Bangalore Genei, India make were used to initiate PCR amplifications. Primers were randomly selected on the basis of GC content and annealing temperature for RAPD-PCR amplifications. After initial screening with all the ten primers, only 3 were used in the final study. They are RAn-1, RAn-5 and RAn-6 with accession numbers AM765825 AM750052 and AM765829, respectively.
The reaction mixture for PCR (30 μL) was composed of 3 μL of 10X Taq polymerase buffer, 1.2 μL of 10 mM dNTP, 1.2 μL of RAPD primer, 0.6 μL Taq DNA polymerase (3 U μL-1), 23.4 μL sterile distilled water and 0.6 μL template DNA. A negative control, without template DNA was included in each round of reactions. After pre-heating for 5 min at 94°C, PCR was run for 45 cycles. It consisted of a 94°C denaturation step (0.45 min), 37°C annealing step (1 min) and 72°C elongation step (1.5 min) in a thermal cycler (Corbet Research, Australia). At the end of the run, a final extension period was appended (72°C, 7 min) and then stored at 4°C until the PCR products were analyzed.
The amplified DNA fragments were separated on 1.2% agarose gel and stained with ethidium bromide. A low range DNA marker was run with each gel (100, 200, 300, 600, 1000, 1500, 2000, 2500 and 3000 bp DNA ladder from Bangalore Genei, Bangalore, India). The amplified pattern was visualized and photographed by Gel Documentation System (Alpha-Innotech, USA).
The RAPD products were scored for the presence and absence of fragments on
the gel photographs and RAPD fragments were compared among the H. fossilis
populations. The RAPD banding patterns were recorded on spreadsheets, which
were used to determine gene diversity, gene flow, number of polymorphic loci
and genetic distance through a construct by an un-weighted pair group method
of arithmetic mean of UPGMA (Nei, 1978) using past (1.
91) software. The Similarity Index (SI) values between the RAPD profiles of
any two individuals on the same gel were calculated using the following formula:
where, NAB is the total number of RAPD bands shared by individuals
A and B and NA is the number of bands scored for each individual,
respectively (Lynch, 1990).
RESULTS AND DISCUSSION
In the present study twelve classical morphometric characters were studied in the two populations of H. fossilis which have been shown in Table 1. The morphometric characteristics did not vary much among the populations from upper lake and lower lake of Bhopal.
DNA from all the 06 samples was extracted using phenol: chloroform:lsoamyl
method (Wu et al., 1995). The extracted DNA of
all the samples was diluted as per the requirements of PCR amplifications. Out
of these, three primers (RAn-1, RAn-5 and RAn-6) generated higher number of
bands (Table 2). The RAPD profile of the bands obtained in
the two populations with primer RAn-05 is shown in the Fig. 1
as a representative photograph. These three primers generated a total of 128
bands in all the 06 individuals out of which 18.75% were polymorphic (Table
2, 3). The UPGMA dendrogram was prepared based on genetic
distance indicating the segregation of the H. fossilis populations collected
from the two reservoirs of Bhopal. The un-weighted dendrogram divided all the
06 individuals in two groups, as observed in the study (Fig. 2).
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 the present study, most of the morphometric characteristics
of fishes were similar and often overlapped with population (Table
||Morphometric measurement of fishes collected from upper and
lower lake, Bhopal
|UL-1, UL-2 and UL-3 are samples from upper lake; LL-I, LL-II
and LL-III are samples from lower lake
||Primer wise pattern of polymorphism between six individuals
||Pattern of polymorphism and uniqueness (4 primers) between
||Random amplified polymorphic DNA fragment patterns generated
using primer 5. 1-3: Fishes of upper lake, 4-6: Fishes of lower lake. M
is the molecular marker of low range DNA ladder
||Phylogenetic tree constructed by distance matrix (Jaccards
These morphometric 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 morphometric analyses were used to discriminate Hilsa populations which
were collected from 9 different sites within Bangladesh (Salini
et al., 2004). They had observed significant differences in allele
frequencies and morphological variations in Hilsa, which may be due to the local
The RAPD markers have been found to have a wide range of applications in gene
mapping, population genetics, molecular evolutionary genetics and plant and
animal breeding (Bardakci and Skibinski, 1994; Ertas
and Seker, 2005). The RAPD technique consists of amplification by PCR, of
random segments of genomic DNA using a single-short primer of arbitrary sequence.
There is no requirement of prior knowledge of the sequence of DNA. Its cost
effectiveness provides an advantage in population genetics studies. The RAPD
technique has been applied to the study of phylogentic relationship in tilapiine
and cichlid species (Bardakci and Skibinski, 1994).
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).
Intra-population 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 Shad from Ganga, Yamuna, Hoogly and Narmada Rivers of India
using RAPD technique (Brahmane et al., 2006).
Garg et al. (2009) studied genetic diversity
of two populations of Mystus vittatus of Madhya Pradesh using RAPD markers
and obtained high polymorphism (64.98%). 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 genetic stock of H. fossilis has not been studied in the reservoirs of Madhya Pradesh. In the present investigation, RAPD analysis has been used to discriminate between the two different populations of H. fossilis of Madhya Pradesh. RAPD fragments observed in the 06 individuals, showed a reasonable degree of genetic diversity within and between the populations. The average percentage of polymorphism was 18.75 as obtained using the 3 primers. The present study may serve as a reference point for future examinations of genetic variations within the populations of fishes which are not only commercially important but also play a significant role in food chain in lentic as well as lotic habitats.
Financial support from the Govt. of Madhya Pradesh is highly appreciated. The authors wish to thank Prof. Pramod K. Verma, Director General and Dr. Rajesh Sharma, Resource Scientist and In-charge CEBT, MPCST for their help and encouragement during the study. The authors also wish to thank Ms. Rashi Parashar for her help during this study.
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