Livestock Research for Rural Development 27 (4) 2015 Guide for preparation of papers LRRD Newsletter

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Genetic diversity of common carp from Shkodra Lake based on microsatellite markers

A Biba, A Hoda, E Vardhami and V Bozg

Agricultural University of Tirana, Albania



Common carp (Cyprinus carpio) of Shkodra Lake is one of the most important fish species. The genetic diversity of common carp in Shkodra Lake was evaluated by the use of five microsatellite loci. A total of 30 individuals were genotyped. The allelic number varied from 18 to 26. Mean observed heterozygosity HO= 0.54 was lower than mean expected heterozygosity HE= 0.92. The population was in Hardy Weinberg equilibrium for none of the loci. All the loci were highly informative, mean PIC value was 0.926. The values of Shannon information index (I) ranged from 2.5-3.3. The mean fixation index over the five loci was 0.421. The bottleneck analyses showed no recent bottleneck.

Key words: expected heterozygosity, fixation index, Hardy Weinberg equilibrium, loci, observed heterozygosity


Shkodra Lake is the largest Lake in Balkan Peninsula, having a high variety of fish species. There are about 60 fish species belonging to 17 families. Cyprinids are one of the most important fish families based on the importance of the species for the daily consume and the large number of species (Shumka et al 2008). Rich in plants and animals it represents an important ecosystem not only for Albania but also at regional aspect. It is a reservoir of biodiversity and a migration route for many species in the region (Shumka et al 2013).

During the repopulation of the Lake there were used also fingerlings of other invasive species (Flloko 2005, Shumka et al 2008), such as Carassius carassius which competes for the habitat of common carp. This situation puts at risk the sustainability of common carp populations (Dhora et al 2013; APAWA and CETI 2007). Hydrolisation and hybridization with improved races have changed the morphometric characteristics of common carp in Shkodra Lake (Shermadhi and Spaho 2013). Another factor that put at risk carp population is overfishing and the increase of illegal fishing during the spawning period (Dhora et al 2013; APAWA and CETI 2007). During the period 2007-2011 the quantity of carp fished grew up 2-3 times normal values the demand for fish is increased with the growth of population, economic level, processing industry, transport (Dhora et al 2013).

Carp of Shkodra Lake has been object of several studies. Golemi et al (2012, 2013) have studied biochemical and hematological parameters of this specie, and the influence of sex in hematological and morphometric parameters of carp. Evaluation of some metabolic and histopathological parameters were used to detect the pollution stress of carp (Aliko et al 2012).  Kolaneci et al (2009) has studied the biological properties of carp population in Shkodra Lake. Shermadhi and Spaho (2013) have studied the morphometric and meristic features variability of carp of three Lakes. Berberi and Shumka (2013, 2014) studied clinical indicators priory to diagnosis of spring viremia of carp and the detection of spring viremia in common carp of Shkodra Lake.

In the current study, five microsatellite markers were used in order to estimate the genetic diversity of Cyprinus carpio for Shkodra Lake.

Material and methods

Fish fins were taken from 30 individuals captured in the Albanian part of Shkodra Lake and were used for the DNA isolation according to standard procedures. Five microsatellite loci (MFW1, MFW6, MFW7, MFW18, MFW9) were analyzed in these individuals. DNA fragments were amplified by polymerase chain reactions (PCR) as described previously (Biba et al 2014). The amplified fragments were separated by vertical electrophoresis in 6% denaturing polyacrylamide gel using 4300 DNA Analyser (LICOR).

Average expected (HE) and observed (HO) heterozigosity values were calculated by Genalex 6.5 program (Smouse and Peakall 2003). The same software was used to evaluate Hardy Weinberg Equilibrium. Polymorphism information content (PIC) values, which is a statistical assessment of informativeness of a marker, were estimated by Cervus software (Marshall 1998). Factorial Correspondence Analysis (FCA) was carried out by Genetix software v4.02 (Belkhir et al 2001). Bottleneck software analyses were used to detect whether carp population of Shkodra Lake has experienced a recent bottleneck, we used Wilcoxon sign-rank test (Piry et al 1999). Wilcoxon test has high power and can be used as few as four polymorphic loci. Mode shift indicator, which discriminates bottlenecked populations from stable populations, was used.

Results and Discussions

The allelic frequencies for the 5 loci are displayed in Figure 1.

Figure 1. Allelic frequencies of five microsatellite loci.

Genetic diversity parameters are shown in Table 1. All markers were highly polymorphic. PIC values for all markers were higher than 0.8, therefore these markers appeared to be highly informative (Botstein et al 1980) and appropriate in genetic diversity studies. A total of 69 alleles were observed at these 5 markers. All markers displayed significant departure from Hardy Weinberg equilibrium, this may be due to non-random matting or natural selection. A common cause of non-random matting is inbreeding. This is supported by fixation index data. Observed heterozygosity (HO) ranged from 0.29 (MFW7) to 0.79 (MFW6) with a mean value of 0.54. Expected heterozygosity (HE) ranged from 0.87 (MFW18) to 0.95 (MFW6) with a mean value of 0.92. High values of expected heterozygosity reflect the presence of variation in this population. Losses in heterozygosity values were published by Lehoczky et al 2005 (HO=0.557; HE=0.700) using 12 microsatellite loci in six Hungarian strains of common carp. Alam and Alam (2012) revealed also heterozygosity loss in three captive strains of Bangladesh common carp analyzed by three microsatellite loci. Fixation index values ranged from 0.17 (MFW6) to 0.69 (MFW7) with a mean value of 0.42 indicating high level of heterozygote deficiency. This can be explained by the presence of null alleles, Wahlund effect, and inbreeding. The values of Shannon information index (I) ranged from 2.5-3.3.

Table 1. Genetic diversity of Cyprinus carpio at five microsatellite loci
Locus N Na Ne I HO HE F PIC Sign (P)
MFW1 27 26.0 17.3 3.0 0.41 0.94 0.57 0.94 <0.001
MFW6 29 33.0 23.0 3.3 0.79 0.95 0.17 0.95 <0.01
MFW7 27 25.0 17.5 3.0 0.29 0.94 0.69 0.94 <0.001
MFW18 27 19.0 8.1 2.5 0.48 0.87 0.45 0.87 <0.001
MFW9 28 18.0 13.6 2.7 0.71 0.92 0.23 0.92 <0.001
Mean 27.6 24.2 15.9 2.93 0.54 0.92 0.42 0.93
SE 0.40 2.70 2.45 0.13 0.09 0.01 0.09    

Number of individuals (N), number of alleles (Na), Effective number of alleles (Ne) Information index (I), Observed heterozygosity (HO), Expected heterozygosity (HE), Fixation index (F),Polymorphism information content (PIC).

Wilcoxon sign-rank test revealed probability values of 0.156 (IAM), 0.812 (TPM) and 0.625 (SMM) which were not significant (p>0.05), so null hypothesis of mutation drift equilibrium was accepted under three models. These results shows that Cyprinus carpio of Shkodra Lake did not experienced a recent bottleneck. The same conclusion was also supported by the mode-shift indicator, which showed a normal L-shape (Figure 2).

Figure 2. Mode shift curve showing lack of bottleneck in C. carpio of Shkodra Lake.

Figure 3. Factorial correspondence analyses between individuals of Cyprinus carpio of Shkodra Lake.

Factorial correspondence analysis (FCA) suggests that the first axis accounted for 5.51%; second 5.26% and third 5.16% of the total variance (Figure 3) showing close genetic relationship between individuals. Only few individuals show higher genetic distance and greater genetic diversity from the other part of the population.

The whole population showed a high deficit of heterozygotes of 42.1%, which supports also a close genetic relationship between sampled individuals displayed by FCA.



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Received 25 January 2015; Accepted 15 February 2015; Published 1 April 2015

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