Genetic Evaluation Of Channa Punctatus Population In Indus River By Using Microsatellite

Channa punctatus, widely known as Murrel or “Shakur,” is a freshwater species native to tropical Africa and southern Asia.

Genetic Evaluation Of Channa Punctatus Population In Indus River By Using Microsatellite

The spotted snakehead, or Channa punctatus, is a species of snakehead fish that is a popular food fish in many parts of Asia and is also used in traditional medicine. However, it is also considered to be an invasive species in some parts of the world, such as the United States.

The freshwater ecosystems are the most endangered water regimes in the world, being devasted by anthropogenic activities such as poor management, habitat fragmentation, and the incorporation of exotic species.

Nowadays, enormous pollution loads in the Indus River are caused by municipal discharge, industrial waste, and agricultural run-off. On the other hand, human interventions, a lack of rehabilitation programs, and inefficient supervision are depleting the genetic diversity of commercially significant fish species.

Freshwater habitats are fragmented as a result of a mix of natural and manmade processes, and their one-dimensionality increases their susceptibility to being detached from two-dimensional forest landscapes.

Fragmentation of species distribution to small, allopatric populations may result from depletion of genetic diversity at the species and within species levels, posing a serious threat to biodiversity’s existence. Many fish species migrations are disrupted by dam construction, interfering with their life cycles. Therefore, natural water systems have lost spawning, breeding, and nursing grounds as a response to hydrological changes and greater human interference in recent years.

Environmental hazards, including climatic change, floods, and human interventions such as pollution, the introduction of exotic species, overfishing, and hydrological alterations, are significant factors that have had diverse impacts on the survival of fish species in recent decades.

Rapid environmental change has a great influence on natural ecosystems and their inhabitants, which pose serious threats to aquatic species by exerting stress like sedimentation, low pH, and changes in salinity and total hardness of water, which lead to vulnerability and extinction.

The pollution adds to the problem by lowering the rate of survival of fish spawn. Siltation caused by deforestation and indiscriminate capture of brood fish during breeding season are two examples of human interventions that have led to the reduction of wild fish populations.

In Pakistan, there is a lack of awareness about genetic issues related to artificial breeding. Numerous private and public fish hatcheries try to protect endemic and cultured species from being genetically degraded.

Channa punctatus, widely known as Murrel or “Shakur,” is a freshwater species native to tropical Africa and southern Asia. Rivers, swamps, rice fields, canals, lakes, and marshes seem to be the most prevalent habitats.

The genus Channa is widely distributed in Asian countries like India, Pakistan, Thailand, Indonesia, Vietnam, Iran, China, Malaysia, and Sri Lanka. In India’s local markets, demand for fish is always greater than supply, and it fetches a good price (INR 60–80/kg) when sold alive, especially in the north and east. Therefore, it is considered a threatened species due to the extra exploitation of natural resources.

Punctatus is known as a ‘lean’ fish because it has very few or no adipose tissues. This fish is famous for its essential amino acids as well as dietary minerals like iron, manganese, calcium, magnesium, and zinc. Moreover, the antioxidant vitamin C is well-known for its powerful anti-stress properties.

Most fish species are unable to produce vitamin C and must rely on external sources to meet their nutritional requirements. Considering the significance of this fish species, the vitamin C requirement for proper growth and survival is fairly modest (both from commercial as well as conservation points of view). So, this fish is considered both economically and aesthetically valuable as a table fish.

Because of its medical and pharmaceutical properties, it is considered the best remedy in the medical field. Additionally, it has great tolerance for adverse water quality and the ability to withstand harsh environments; it also has a high market price.

As it is an air-breathing fish, it has been recognised as a potential candidate species for aquaculture in derelict and swampy water. Along with its taste, low intramuscular spines, and high protein content, it has a high consumer preference.

Many elements influence the culture’s success, the most important of which is seed availability. That’s why it concerns a significant species for culturing as well as research.

Our Objectives.

  • To evaluate the extent of genetic diversity in the natural population of Channa punctatus
  • To assess the current genetic structure in the riverine population of C. punctatus by using microsatellite markers


Numerous anthropogenic activities have consistently posed a serious threat to the genetic integrity of fish populations. Global biodiversity is being eroded as a result of these genetic diversity losses.

The proposed study was conducted to genetically analyze Channa punctatus populations in the Indus River collected from five different sites, viz., Chashma Barrage, Taunsa Barrage, Jinnah Barrage, Ghazi Ghat, and Guddu Barrage. Samples were brought to the Aquaculture Biotechnology Lab, University of Agriculture, Faisalabad, while being stored on crushed ice.

DNA was extracted using standard phenol/chloroform DNA isolation methods, and the quality and quantity of the isolated DNA were examined by agarose gel electrophoresis and nanodrops, respectively. The genomic DNA of C. punctatus was PCR-amplified by cross-species amplification of C. argus, and PCR products were further resolved on 5% non-denaturing PAGE.

The data were analysed using a variety of software programmes, such as FSTAT, POPGENE, and TFPGA. A moderate level of genetic diversity was recorded in the studied populations, as the number of alleles on each locus ranged from 2.0 to 7.0 with an average of 3.00 to 4.60. Average observed and expected heterozygosities ranged from 0.580 to 0.680 and 0.560 to 0.759, respectively.

The average fish values were -0.088 to 0.017 in all of the examined populations of C. punctatus. Genetic distance among populations showed significant variation (P<0.05), with a maximum value of genetic distance of 0.2053 in GB-JB and a minimum of 0.0194 between GG-TB.

The AMOVA revealed that in populations of C. punctatus, the majority of the variation was found within individuals. Two large clusters, A and B, were formed by the UPGMA dendrogram: one cluster consisted of CB, TB, and GG, while the other consisted of GB and JB. The results of this study will be helpful for Pakistan’s native fish species to be monitored, conserved, and restored to achieve sustainable fisheries goals.


A sample of Channa punctatus was taken from five selective sites in different regions. A total of 125 were collected, and 25 were from each site. Muscle tissue was taken from each specimen for DNA extraction.

DNA isolation and quantification

Genetic DNA isolation consists of the following steps:

Analysis of DNA

A nanodrop was used to assess the quantity of isolated DNA. A 1 µL DNA sample was used to check the concentration of the isolated DNA.

The concentration of isolated DNA was adjusted to 50 g/µl for PCR by mixing a stock solution of DNA with nuclease-free water. Calculation was done by using the formula: C1 V1=C2 V2, C1 = stock DNA solution, V1 = volume of DNA required, C2 = diluted DNA concentration, and V2 = total volume of diluted solution required.

Electrophoresis and visualisation for the separation of amplified products

The amplified XDNA was isolated on a 5% non-denaturing PAG with 19:1 acrylamide:bis-acrylamide and viewed by silver staining. Electrophoresis was conducted using the sequencing gel electrophoresis method.

Using an alpha-image reader, the resulting gel was visualised by autoradiography. Alleles size was calculated concerning the ladder sequence.

Data Analysis

Probable genotype errors such as sturting bands, null alleles, and large allele dropouts were analysed using MICRO-CHECKER software.


Observing genetic diversity and genetic structure analysis of the natural population, all five microsatellite markers were found to be polymorphic.

The average allele frequency range (0.007–0.527) and allele size (255–277) varied in form and base pairs.

The heterozygosity level was found to be moderate in all examined wild populations of C. punctatus in the present study. The average values of observed heterozygosity (H0) were measured and ranged from 0.580 to 0.680.

Microsatellite analysis using the structure grouping algorithm showed the presence of two separate genetic clusters in the collected C. punctatus populations.