Investigating allelic polymorphisms in Cyprinus carpio (common carp).

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Abstract

Genetic variations play important roles in the survival of fish as it provides the potential to adapt to fluctuating environmental conditions. Genetic diversity is being deteriorated by various environmental and anthropogenic activities including pollution, over exploitation of fish stock and habitat loss. To estimate allelic polymorphism in Cyprinus carpio, sampling will be done from river Chenab (Pakistan). At least 30 samples of fish will be taken to University of Agriculture, Faisalabad. Genomic DNA will be extracted from muscle tissue using different methods and techniques. Five microsatellite loci (MFW-1, MFW-2, MFW-17, Barb-22, Bgon-54) will be used to access genetic variation and the mean values of observed and expected heterozygosity will be calculated. The Hardy-Weinberg theorem will be used to investigate the allelic frequency and deviation from this equilibrium will designate genetic diversity in populations. Data that will be obtained from this research work will analyzed by using different software. Studies on the allelic polymorphism of C. carpio would be helpful in lessening the genetic concerns relating to C. carpio populations.

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Genetic diversity in specie assures the survival of specie by providing the capability to combat with varying environmental conditions. Genetic drift, inbreeding, unintended selection of broodstock, artificial selection are the various factors which influence genetic variation in a specie by carrying random variations in gene frequency (Mignon-Grasteauet et al., 2005).

The common measure of genetic diversity includes allele frequency, heterozygosity, population variation, linkage disequilibrium, inbreeding coefficient. The Hardy-Weinberg theorem is used to investigate the allelic frequency and deviation from this equilibrium designates genetic diversity in populations (Okumus and Ciftei, 2003).

Molecular markers are important tools use to study the levels and pattern of genetic diversity and have been used to study the genetic diversity of numerous farmed fishes. Only recently mitochondrial DNA (Gross et al., 2002), AFLP (David et al., 2001), RAPD and microsatellite markers (Bartfai et al., 2003) are being used.

DNA microsatellites are the most useful molecular markers that are extensively and efficiently used in the estimation of the genetic diversity of various organisms. These markers are highly polymorphic tandem arrays of short nucleotide sequences distributed throughout the genome of eukaryotes and have been used effectively for monitoring genetic diversity in different common carp strains (David et al., 2001; Lehoczky et al., 2002; Bartfai et al., 2003; Kohlmann et al., 2003). They are inherited in Mendialian fashion.Because of high level of polymorphism.co-dominance, high mutation rate and easy analysis using PCR, microsatellite marks are used to evaluate the genetic diversity of numerous food fishes species such as salmon (Norris et al., 1999), rainbow trout (Thrower et al., 2004) and tilapia (Rutten et al., 2004).

These markers can be utilized in aquaculture due to their unique features including high level of polymorphism, fast detection protocol and comparatively small size. There markers can be utilized in aquaculture for percentage identification, management of brood stock, marker supported breeding suites, individual identification and to construct genetic linkage map (Wang et al., 2009).

Common carp (Cyprinus carpio) belongs to largest fresh water teleost family Cyprinidae. Common carps are native to Europe but have been widely introduced and are now established everywhere except poles and northern Asia. (froesa et al., 2002).

Wild common carp are extremely vulnerable or already vanished in many natural areas due to high level of human pressure (overfishing), habitat loss, pollution, degradation of breeding grounds and hybridization with domestic carp (Kohlmann et al., 2005)

The total world annual production of common carp in 2003 has been estimated to be 3,239,712 tons (FAO, 2005). Now a day, people do not like aquiculture yields, they favor the wild ones. This makes the wild carps more expensive, and more wild carp are caught from rivers, which results in the lessening of wild carp resources. The proportion of wild carps is also reduced in the total aquatic product. The loss of genetic resources and genetic decline is increasing. Considering the genetic variations of the wild common carp population is important for genetic resource protection, protecting genes of significant characteristics, and limiting the genetic decline (Dayu Li et al., 2001).

The present area of study is river Chenab which starts from himachal Pradesh (india) and enters into Pakistan near diwara district of Sialkot (Punjab). In the Punjab, this river runs through Sargodha, Gujarat and Gujranwala districts. Total length of river is 960km with an area of 41656km. Head Marala, Qadirabad, Khanki and Head Trimmu are the important water reservoir Studies on the allelic polymorphism of C. carpio would be helpful in lessening the genetic concerns pertaining to C. carpio (Siddiqi et al., 2004).

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REVIEW OF LITERATURE

Kohlmann and Kersten (1999) carried out an electrophoretic study of the allozymes of different strains of common carp and reported that the average number of allozyme alleles per locus was 1.4 to 1.9, the mean H0 was 0.112 to 0.256 and the percentage of polymorphic loci was 25 to 50.

J.F. DesÕignes et al, (2001) conducted an experiment to study genetic variability of cultured stock of common carp which contain six strains from two French regions Dombes and Forez and five strains from the Czech Republic retained in the Research Center of Vodnany, using two molecular markers allozyme and microsatellites. Using allozyme, he observed that heterozygosity for French strain (0.003 to 0.029) was considerably lower than that observed in Czech strains (0.026 to 0.058), and the average number of alleles per microsatellite locus were greater for French strains than for Czech strains correspondingly, 6.2 and 4.2. He concluded that the genetic variability using microsatellite was significantly greater than that for allozyme.

Kohlmann et al, (2005) studied the micro satellite based genetic variability and differentiation of domesticated, wild and feral common carp populations and concluded that four micro satellite loci displayed a high genetic variability. A total of 143 alleles were recorded over al loci ranging from 27 at MFW28 to 47 at MFW7. The reduction in genetic variability in domestic and wild cap was more pronounced in microsatellite loci than at allozyme loci examined in previous studies (Kohlmann and Kersten ,1999).

I. Lehoczky et al, (2005) studied the genetic description of two wild strain (Dunai and Tiszai) initiating from two major river systems, Danube and Tisza of Hungary. He used mitochondrial NADH dehydrogenase (ND-3/4 and ND 5/6) genes (PCR- RFLP) and four microsatellite loci (MFW1, MFW6, MFW7, MFW28). He analyzed PCR-RFLP using restriction enzyme, at ND-3/4 (HinfI, AluI, HpaII and TaqI), and at ND-5/6 (BsuRI, Eco471) that mitochondrial genes did not provide evidence for mingling of Asian and European carp in above examined carp strains while the microsatellite data showed that the average number of alleles were significantly greater in Tiszai (11.25) than that of Dunai (8.75) with mean observed hetrozygosity of 0.784 and 0.942 and mean expected hetrozygosity of 0.850 and 0.831 respectively with significant deviation from Hardy- Weinberg equilibrium at 3 loci in Tiszai and at all 4 loci in Dunai population.

Mondol et al, (2006) studied characterization of different strain of common carp in Bangladesh using microsatellite DNA markers, concluded that all the microsatellite loci subjected to PCR amplification were found to be polymorphic .The MFW15 locus had the highest number of alleles (11) The average number of alleles across all loci was the highest (7.40) in the red carp. The observed (Ho) in the koi carp was t highest (0.76) than that of red, mirror and scaled carp (0.71),(0.69), (0.5) respectively. He concluded that koi carp stock displayed an excess of heterozygosity at most of the loci. The sizes of the alleles ranged from 124 to 264. No strain-specific private allele was present for any strain at any of the five loci.

B.T. Thai et al, (2006) studied the genetic variation of twenty strain of common carp with 968 representatives screened for variation in mitochondrial control region fragment by using direct DNA sequencing and SSCP (single strand confirmation polymorphism) analysis. Samples of carp were collected from Japan, Hungary, China and Indonesia for comparison. DNA sequencing shown that Vietnam common carps have high haplotype but low nucleotide diversity representing a mixture of indigenous and introduced carp strains while the SSCP analysis revealed eight haplotypes distinguishing Indonesians, Hungarian and Vietnams strains.

D. MemiÅŸ, K. Kohlmann (2006) conducted experiments on genetic variability of common carp in three lakes of Turkey (Sapanca and Iznik) using microsatellite loci ,restriction fragment length polymorphism (RFLP) of mitochondrial ND-3/4 and ND-5/6 gene regions and concluded that genetic variation between Turkish wild carp were considerably higher. PCR-RFLP analysis showed five mixed haplotypes. One of them was similar to European /Asian haplotypes to above examined populations while other four were different in only one or two restriction enzyme fragment patterns.

M. Hulak et al, (2010) studied molecular genetic variation in common carp strains stocked in Czach Rpublic by microsatellite analysis of 10 loci. To this he conducted experiment on 11 carp strains two from Germany (Scheuermn and Glinzinig mirror carp), France (Forez and Domber scaly carp), Spain (Ebro river) and one from Amur river and observed average heterozygosity from 0.584 to 0.700 with average number of alleles per population ranged from 5.0 to 9.8. he concluded a significant deviation from Hardy-Weinberg equilibrium (PË‚ 0.05).

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T. Tomljanović et al, (2013) studied common carp population in Croatia using 15 microsatellite and 243 samples, five from wild and hatchery respectively. He recorded a total number of 148 alleles but the average numbers of alleles per locus were considerably low. He concluded that microsatellite markers are a powerful tool in estimating the various genetic conditions of common carp in Croatia. He suggested that the lessening of allelic diversity might be due to irregular blockage effects during breeding courses.

MATERIALS AND METHODS

Sample collection

Total 30 samples of C. carpio including fingering and adults will be collected from river Chenab, Punjab province of Pakistan. For DNA extraction dorsal muscle tissue will be excised at the sampling place and will be kept in polythene bags for identification. The samples will be than immediately placed in crushed iceboxes at temperature -20°C to transport them to the laboratory.

DNA extraction and Quantification

The scales, spines and fins will be removed and genomic DNA will be extracted from the dorsal muscle tissue following the Yue and Orban (2005) technique with slight amendments. Twenty samples will be selected for DNA isolation. DNA isolation procedure comprising tissue lysis and DNA washing will be done by using different chemical reagents (EDTA, HCl, NaCl, SDS etc). Quantification of DNA will be done using agarose gel solution, IX TAE buffer, Bromophenol blue etc.

DNA analysis by Spectrophotometer

The quality and concentration of isolated DNA samples will be aasessed by the use of UV spectrophotometry.

Microsatellite loci analysis

PCR will be used to amplify genomic DNA of isolated microsatellite loci by different primers (MFW-1, MFW-2, MFW-17, Barb-22, Bgon-54), which will be purchased from company gene bank.

Gel electrophoresis analysis

Polyacrylamide gel electrophoresis (PAGE) will be run for electrophoretic resolution.

Data analysis

Several parameters will be analyzed by using different software, and the allele frequency, allelic abundance and observed and expected heterozygosity will be calculated through statistical analysis.

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