RAPD-PCR Based Biomarker Study in Fish Species (Family: <em>Cyprinidae</em>) of Madhya Pradesh, India

Research Article

Austin J Mol & Cell Biol. 2014;1(1): 1003.

RAPD-PCR Based Biomarker Study in Fish Species (Family: Cyprinidae) of Madhya Pradesh, India

Neekhra B¹, Mansoori AA¹, Verma S¹, Koiri RK² and Jain SK¹*

1Department of Biotechnology, Dr. Hari Singh Gour Central University, India

2Department of Zoology, Dr. Hari Singh Gour Central University, India

*Corresponding author: Jain SK, Department of Biotechnology, School of Biological Sciences, Dr. H. S. Gour Central University, Sagar – 470003, India,

Received: October 14, 2014; Accepted: December 01, 2014; Published: December 31, 2014


Random Amplified Polymorphic DNA (RAPD) analysis was conducted for molecular identification of most commonly occurring fish species in Central India. In the study, male and female species of Labeo rohita, Catla catla and Cirrhina mrigala (family: Cyprinidae) were randomly collected from various locations of central Indian region. For polymerase chain reaction, ten decanucleotide primers (RAn primer series) were used. On the basis of interpretability, simplicity and reproducibility, six random primers RAn 1 (GATGACCGCC), RAn 3 (GGCACGTAAC), RAn 4 (GGCATGACCT), RAn 5 (GGGTAACGCC), RAn 9 (GTGCCAAATG) and RAn11 (GTGCCCGTTA) were considered for genetic differentiation and band analysis. We observed varied size of amplified products depending upon the sequence of random primers and genotypes used. A total of 55 discrete amplified products were obtained (size 200 to 2000 bp approximately). Out of 55 products, 29 were species specific markers indicating high level polymorphism among species. The highest and lowest number of RAPD bands detected for primers RAn 5 and RAn 9 was 12 and 6 respectively. 16 bands were most relevant as found common in all three species of family: Cyprinidae. Highest monomorphism observed for C. catla and C. mrigala (5 bands) and lowest for L. rohita & C. mrigala (1 band). Similar band patterns were obtained for both male and female of all three fish species showing 100 percent sexual genetic similarity. The diverse nature of DNA bands indicated the genetic distance between fish species and presence of common bands attributed to an evolutionary relationship. Pattern of species specific unique bands observed might be useful tools for molecular identification. Our study re-establishes the importance of RAPD-PCR technique for molecular taxonomy studies in diverse fish species.

Keywords: Genetic differentiation; Polymorphism; RAPD- PCR; Random primers


India has about 4.4 percent of the global fish production; this sector contributes to 1.1 percent of the GDP and 4.7 percent of the agricultural GDP. The total fish production of India (2010-11) is around 7.50 million tones including 4.50 million tones of inland fish and 3.00 million tones of marine fish [1]. The application of genetics in aquaculture and fisheries resource management has been very limited as compared to agriculture, forestry and animal husbandry.

Information on the molecular structure of fish species is useful for optimizing identification of stocks, stock enhancement, breeding programs, management for sustainable yield and preservation of genetic diversity [2-5].

Molecular techniques based on DNA sequence polymorphism are now used in population genetics studies, systematic and molecular taxonomy to get an answer to systematic related problems. Molecular techniques played an important role to understand the basis of polymorphism of a species, species diagnostics and population differentiation [6].

Within the last decade, scientific progression has increasingly supported the use of molecular techniques in determining population diversity. Many molecular techniques are now available, which allow ecologists and evolutionary biologists to determine the genetic architecture of a wide variety of closely related individuals. The discovery of the PCR had a major impact on the research of eukaryotic genomes and contributed to the development and application of various DNA markers [7-9].

RAPDs are particularly useful to study the genetic structure of populations because they reveal polymorphisms in non-coding regions of the genome. Random primers produce RAPDs that have been used extensively as molecular markers [10-11]. RAPDs also have the advantage that no prior knowledge of the genome is necessary for successful application [12-13].

The RAPD markers method has been reported to be an efficient tool to differentiate geographically and genetically isolated population, and has been used to verify the existence of population of species that have arisen either through genetic selection under different environmental conditions or as a result of genetic drift [14]. During the last few years, RAPD-PCR has been shown wide range of applications in fisheries and poultry [15-21].

In view of the above facts, the present study was aimed to develop RAPD fingerprinting in Labeo rohita, Catla catla and Cirrhina mrigala collected from Sagar region, Madhya Pradesh and also aimed to generate species-specific RAPD marker as a reference database for species recognition and characterization. Thus, the investigation will provide valuable tool in the form of diagnostic markers for fisheries conservation and management of these species.

Material and Methods

Sample collection

Three species Labeo rohita, Catla catla and Cirrhina mrigala (male and female) were collected from local rivers and lakes of Sagar region and preserved at -20°C. The most commonly used sources of fish DNA are scales, fin and muscles tissue. A rapid DNA extraction method developed by using modified lysis buffer that require about 2 hours duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery [22].

DNA extraction

Approximately, 50 mg of scales were taken from each species and dried on a filter paper. The scales were then cut into small pieces and placed in a 2 ml-Eppendorf tube containing 940 μl lysis buffer (200mM Tris-HCl, pH 8.0; 100mM EDTA, pH 8.0; 250 mM NaCl), 30 μl proteinase K (10 mg/ml) and 30 μl 20 percent SDS. The content in the tubes were incubated at 48°C for 45-50 min in a water bath. After incubation, 500 μl volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to the tube containing lysed scales cells. The contents were then mixed properly by gently inverting the tube for 10 min to precipitate the proteins and other part of the nucleic acids. The tube was then rotated for 10 min at 9,200 g. The top aqueous layer was transferred to a new 1.5 ml-Eppendorf tube, leaving interface and lower phase. The DNA was precipitated by adding equal volume of isopropanol and 0.2 volumes of 10 M ammonium acetate and inverting the tubes gently several times. The precipitated DNA was then pelleted by centrifugation at 13,200 g for 10 min. The supernatant was removed by pouring out gently, taking care to avoid loss of DNA pellet. The pellet was then washed briefly in 500 μl chilled 70 percent ethanol, air-dried and resuspended in 200 μl sterile water/ TE buffer.

After ensuring complete solubility of DNA, the purity factor (A260/A280 nm) was measured by UV spectrophotometer (Cole Parmer Ins. Company, US) and its integrity was checked by loading 10 μl DNA preparation (2 μl extracted DNA, 2 μl dye and 6 μl sterile water) on 0.8 percent agarose gel and stained with ethidium bromide. The extracted DNA samples were then stored at -20°C till their further use. These DNAs were used as templates in a PCR based search for producing RAPD markers.

DNA amplification by PCR

Prior to the experimentation pilot study was performed in 10 fishes of each species (both female and male). The data thus observed showed similarity in banding pattern (considering all bands) and therefore only one male and female from each species considered during experimentation.

The DNA was amplified by using 50 μl of reaction mixture containing sterile water 39.0 μl, 10X Taq Buffer A 5.0 μl, 10mM dNTP mix 2.0 μl, RAPD primer 2.0 μl (RAn series supplied by Bangalore genei company), DNA template (10ng/μl) 1.0 μl, Taq DNA polymerase (3 U/μl) 1.0 μl. The amplification was carried out in thermal-cycler (Eppendorf pro) under the PCR conditions were predenaturation at 94°C for 5 min followed by two loops. First loop of 10 cycles of denaturation at 94°C for 45 sec, annealing at 35 °C for 1 min and extension at 72 °C for 1.5 min and, second loop of 40 cycles of denaturation at 94 °C for 45 sec, annealing at 37 °C for 45 sec and extension at 72 °C for 1 min. The final extension was performed at 72 °C for 10 min. On the basis of results of initial RAPD analysis on pooled DNA, 6 out of 10 primers were chosen for further analysis, based on band pattern quality, reproducibility and the presence of diagnostic bands. Finally the RAPD-PCR was done using six primers (Table 1).