Assessment of genetic variability in common whitefish from the catchment area of the Oder river using microsatellite markers

Common whitefish ( Coregonus maraena ) in Poland belongs to the endangered species. The degradation of the environment causes common whitefish to lose its natural reproduction sites. The natural genetic structure of whitefish has been compromised by anthropogenetic activities involving eutrophication, river regulation, the introduction of non-native species and as well as excessive exploitation of the species. The genetic variability of common whitefish ( Coregonus maraena ) from 2 sites: Pomeranian Bay and the lower Oder river, was assessed using microsatellite markers. A total of 45 caught individuals were analysed (26 from Pomeranian Bay and 19 from the Oder river). Polymorphism at nine loci, Str60INRA, Str73INRA, Strutta 12, OmyFgt1TUF, Str85INRA, Str591INRA, Ssa85, Ssa197, T3-13 was assessed. The results indicated that all the investigated populations showed a high level of genetic variability. The level of genetic variability was determined using the F ST parameter and was high investigated populations (0.215). Microsatellite analysis demonstrated a higher observed heterozygosity as compared with the expected heterozygosity in all the investigated populations. The F IS coefficient values below zero in all the investigated populations of common whitefish indicate the excess of heterozygotes. The high number of heterozygotes may be related with a more intense influx of genes from outside of the local population. The study demonstrated that microsatellite markers (SSR) are very useful in the assessment of the genetic variability of common whitefish ( Coregonus maraena ). Our results characterize the selected populations of whitefish and may be useful for further research on this endangered species.


Introduction
Common whitefish (Coregonus maraena) in Poland belongs to the endangered species (Witkowski et al., 2009). Natural populations of this species in Poland occur in Pomeranian Bay and Lake Łebsko (Heese, 1999). Moreover, common whitefish inhabits reservoirs and coastal waters of the Baltic Sea, as well as lakes, e.g., Lake Miedwie and Lake Wigry (Szczerbowski, 2000). The species inhabits clean, cool and well-aerated water. The degradation of the environment causes common whitefish to lose its natural reproduction sites. In the recent years, the number of Polish lakes inhabited by common whitefish has decreased significantly, which is caused by the deterioration of spawning sites (Wilkonska, Zuromska, 1982), as well as excessive exploitation of the species (Witkowski et al., 2009). Currently, large-scale species restoration programmes are being carried out in many lakes. Mass introduction of the fish has been conducted without any preliminary identification of individuals (Szczerbowski, 2000). Introducing closely related individuals derived from a small number of spawners may lead to the impoverishment of the gene pool (Fraser, 2008). However, the restoration of endangered species should be accompanied by genetic monitoring (Foop-Bayat, Wiśniewska, 2010). Such studies are conducted in common whitefish inhabiting Lake Łebsko . Common whitefish observed in the Oder mouth constitutes a large stable population used as a source for introductions. Therefore, it is vital to perform genetic analysis of the species, which will permit a more effective fisheries management. The aim of this study was to assess the genetic variability of common whitefish (Coregonus maraena) from 2 sites, i.e. Pomeranian Bay and the lower Oder river, using microsatellite markers.

Research subject
The analysis was performed in common whitefish from 2 sites: Pomeranian Bay and the lower Oder river (Figure 1), in which the fish putatively form a local population. A total of 45 caught individuals were analysed (26 from Pomeranian Bay and 19 from the Oder river). The fish were weighed and measured, and samples of muscle tissue were taken. The samples were subsequently frozen at -70°C.

Isolation of genomic DNA
DNA was extracted from 0.2 g muscle tissue taken from every caught individual of the investigated fish population. The material was placed in 1.5 ml tubes and 1 ml extraction buffer (100 mM Tris-HCl, 200 mM NaCl, 0.2% SDS, 5 mM EDTA and 100 μg/ml proteinase K) was added. The mixture was incubated at 55°C for 12 h and then centrifuged at 6000 × g for 15 min. The supernatant was transferred into a new tube and 700 μl isopropanol was added. The mixtures were centrifuged again at 6000 × g for 15 min. The supernatant was discarded and the remaining pellet was resuspended in 400 μl 70% EtOH. The mixtures were centrifuged again at 6000 × g for 5 min, the alcohol was discarded and the samples were dried. The pellet was dissolved in 20 μl TE buffer. The extracts were stored at -20°C. The quantity and the purity of DNA was determined using the BioRad SmartSpec TM Plus spectrophotometer.

Statistical analysis of the results
The results were stored on a BioRad gel documentation system and analysed using the Quantity-One® software (BioRad, USA). The number of alleles per locus (Na) and the number of effective alleles per locus (Ne) were calculated for each investigated population and for all populations at the same time. Genetic information was determined for nine SSR loci in 2 populations using the following indices: number of private alleles per population (Np), Shannon diversity index (I) (Shannon, Weaver, 1949), observed heterozygosity (H O ), expected heterozygosity (He), unbiased expected heterozygosity (uHe), inbreeding coefficient (F IS ), fixation index (F ST ). Variability per locus was measured using the coefficient of Polymorphic Information Content (PIC) (Anderson et al., 1993). where: p -band frequency.
The genetic similarity between populations was expressed using Nei's genetic distance (Nei et al., 1983). All calculations were done with GenStat 15th Edition and GenAlEx v. 6.5b4.

Results
The investigated populations were characterized by a high level of genetic variability (Tables 2 and 3). In all of them, the mean number of alleles per locus (Na) was higher than or equal to the mean number of effective alleles per locus (Ne) ( Table 3). Shannon diversity index (I) was insignificantly lower in the lower Oder population compared with the other investigated population. The observed heterozygosity (Ho) was lower than the expected heterozygosity (He) in all populations and most heterozygotes were observed in the Oder river population, while the least were observed in the Pomeranian Bay population (Tables 2 and 3). Private alleles were observed exclusively in common whitefish inhabiting Pomeranian Bay (Table 3). Statistical analyses revealed that the investigated whitefish populations are characterized by inbreeding coefficient (F IS ) below zero, which indicates a high excess of heterozygotes (Tables 2 and 3). The coefficient of Polymorphic Information Content (PIC) values for each of the SSR (Simple Sequence Repeats) primers ranged from 0.47 to 0.90. The mean value of this index was 0.74 (Table 3). The observed heterozygosity values generated by each SSR primer were between 0 and 1, while the values of expected heterozygosity ranged from 0 to 0.53 (Table 3). The level of genetic variability between the populations was determined using the F ST parameter. The genetic distance between the investigated whitefish populations is great (0.215) ( Table 4). The percentage of polymorphic loci was identical in the two investigated populations (66.67%).

Discussion
In the region of the Baltic Sea, the reduction of the common whitefish population is a result of intensive fishing and environmental pollution. The rapid loss of genetic diversity leads to decreased adaptation capabilities and an increased risk of extinction of the species (Frankel, Soul, 1981;Frankham, 1995). If restoration of an endangered species is performed, genetic monitoring should be conducted in parallel to avoid changes in the genetic structure and the impoverishment of the gene pool of the population (Fraser, 2008;Fopp-Bayat, Wiśniewska, 2010). The morphological and genetic variability of Coregonus maraena was assessed using microsatellite sequences Lu, Bernatchez, 1999;Østbye et al., 2004;Hansen et al., 2008) and mitochondrial DNA (Kohlmann et al., 2007;Kempter et al., 2010).
In this study, polymorphisms among common whitefish populations were determined using the microsatellite markers, successfully employed in the assessment of genetic variability and the degree of similarity between fish species (Fopp-Bayat, Säis et al., 2008;Winkler, Weiss, 2008;Dierking et al., 2014).
The values of expected heterozygosity (He), obtained by employing microsatellite sequences in the study of Coregonus maraena populations, ranged from 0.308 to 0.332. The level of heterozygosity of the investigated populations of common whitefish is lower than in the Alpine populations from Austria (He = 0.37 -0.95) (Winkler, Weiss, 2008) and three naturally reproductively isolated whitefish taxa in Germany (He = 0.66 -0.76) (Dierking et al., 2014), but similar to that of Norwegian populations (Østbye et al., 2004). The high number of heterozygotes may be related with a more intense influx of genes from outside of the local population. Various studies indicate that many fish populations, such as Culter erythropterus (Wang et al., 2007), Engraulis encrasicolus (Zarraonaindia et al., 2009), or the investigated Coregonus lavaretus (McCairns et al., 2012), face the problem of a reduced frequency of heterozygotes due to inbreeding (O'Reilly et al., 1996). It is therefore a very positive signal that no such phenomenon is observed in the two investigated populations of common whitefish. Surprising is the fact that the presence of private alleles (Np), occurring exclusively in a given population, was observed only in the common  (Douglas et al., 1999), or those of the populations from Norwegian lakes, ranging from 0 to 0.0143 (Østbye et al., 2004), are examples of this phenomenon. The high level of genetic variability retained by the investigated common whitefish populations indicates that despite the significant reduction of the number of individuals, no effect of genetic drift has occurred. As a result of the long-term effect of the process, a reduction in the intra-population variability occurs along with an increase in the inter-population variability.
The conducted study demonstrated a high genetic variability between the analysed common whitefish populations, which may be explained by the mass introduction of the species into waterbodies without proper identification (Szczerbowski, 2000). It is even hypothesized that finding a pure form of common whitefish is very unlikely (Witkowski et al., 2009).
Continuous genetic monitoring is necessary in the process of renewal of endangered species in order to prevent the disruption of the genetic structure of the population (Fopp-Bayat, 2010). The need to know and to characterize this structure as many populations also indicate other researchers (Pamminger-Lahnsteiner et al., 2009).
Our results characterize the selected populations of whitefish and may be useful for further research on this species.