Genetic Variability in European Populations of Coregonus Lavaretus (L.): an Assessment Based on Mitochondrial ND-1 Gene Haplotypes
Total Page：16
File Type：pdf, Size：1020Kb
Arch. Pol. Fish. (2010) 18: 197-204 DOI 10.2478/v10086-010-0023-y RESEARCH ARTICLE Genetic variability in European populations of Coregonus lavaretus (L.): an assessment based on mitochondrial ND-1 gene haplotypes Received – 27 July 2010/Accepted – 20 October 2010. Published online: 30 December 2010; ©Inland Fisheries Institute in Olsztyn, Poland Jolanta Kempter, Klaus Kohlmann, Remigiusz Panicz, Jacek Sadowski, S³awomir Keszka Abstract. The genetic variability of whitefish, Coregonus Introduction lavaretus (L.), was studied based on 114 individuals from nine populations inhabiting Polish lakes, including the Szczecin Lagoon, and from one population each from lakes in Due to the necessity of exploring the culture poten- Austria and Switzerland. Differences within and among tial of fish species novel to aquaculture as well as at- populations were assessed with mitochondrial ND-1 gene tempts to implement the principles of responsible sequences that were PCR amplified and digested with ten fisheries, including in inland fisheries, scientific in- restriction enzymes. The ten composite haplotypes obtained terest focuses on fish species which are either of great were sequenced and analyzed with MEGA4 software. The highest intrapopulation variability was noted in the whitefish economic importance or promise good market prod- populations inhabiting lakes Iñsko, Miedwie, Marianowo, ucts. Coregonid species, particularly whitefish, Wisola, Œremskie, Morzycko, the Szczecin Lagoon, and Lake Coregonus lavaretus, (L.), is one such species. Since Lucerne, which possessed from two to five composite responsible aquaculture and sustainable fisheries haplotypes. In contrast, populations inhabiting lakes Woœwin, management both require knowledge of the genetic Czarne, and Traunsee were fixed for the most common status of the species being managed, comparative ge- haplotype H2. netic research makes it possible to set up a “gene Keywords: Coregonus lavaretus, PCR-RFLP, ND-1, bank” in the form of information on interpopulation haplotype variability, mtDNA differences and allows assessing the genetic purity of studied populations. The artificial reproduction of J. Kempter [+], R. Panicz, J. Sadowski Department of Aquaculture whitefish resulted in the emergence of whitefish x West Pomeranian University of Technology peled, Coregonus peled (Gmelin) and whitefish x ven- K. Królewicza 4, 71-550 Szczecin, Poland dace Coregonus albula (L.) hybrids. Therefore, the Tel. +48 91 4496664; e-mail: [email protected] practical importance of studying the genomes of po- K. Kohlmann tential breeding stocks is of particular significance. In Department of Ecophysiology and Aquaculture addition, the whitefish is known to have formed pop- Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany ulations of great economic or historic value. One ex- ample is furnished by Lake Miedwie whitefish which S. Keszka for years has been a source of stocking material for Department of Fish Systematics West Pomeranian University of Technology, Szczecin, Poland numerous European lakes. Moreover, if the historical evidence is to be believed, Lake Miedwie whitefish Unauthenticated Download Date | 12/16/15 2:05 AM 198 Jolanta Kempter et al. was an ancestor of whitefish that made their way to Woœwin, Miedwie, Marianowo, Wisola, Czarne Japan (Toshikazu and Tetsuro 2004). (Drawieñski National Park), Œremskie, Morzycko), Coregonids, including whitefish, are particularly the Szczecin Lagoon, and one lake each in Austria frequently subjects of studies on genetic variability. (Traunsee) and Switzerland (Lucerne). The numbers The intense interest in this aspect of the whitefish in Figure 1 denote the number of samples collected stems from its high interpopulation variability, and its in each country. ability to form numerous varieties and races differing in morphology and/or behavior. This raises the ques- tion of whether these differences are reflected in genotypic variability. Thus far, the literature dealing with whitefish has identified numerous varieties and sub-species differing in mouth structure, body shape, and the number of gill rakers, among others (Szczerbowski 1969). Numerous studies have also ad- dressed whitefish genetic variability by examining mi- tochondrial DNA (Bernatchez et al. 1991, Bernatchez and Dodson 1994, Brzuzan 1998, 2000, Brzuzan et al. 1998) and microsatellite markers (Hansen et al. 1999, 2008, Winkler and Weiss 2008). An earlier paper by Kohlmann et al. (2007) fo- cused on the variability of the mitochondrial ND-1 gene in whitefish caught in Poland, primarily in West- Figure 1. Origin of samples used in this study and the number of ern Pomerania. The present study broadens the scope samples collected in each country. of the previous study by extending the sample size from some Polish lakes and by including samples ob- DNA from these samples was isolated using the tained from two European countries with whitefish E.Z.N.A. Tissue DNA Mini Kit (Peqlab Biotechno- populations that have been known for years. Analyses logie, Germany). The isolated DNA was subse- of their intrapopulation variability will make it possi- quently used as a template for the amplification of ble to determine the degree of affinity between these the mitochondrial ND-1 gene region (2012 bp) using populations and to estimate the genetic homogeneity a pair of primers described by Nielsen et al. (1998): of populations so in the future they can serve as a po- Forward: 5’ GCC TCG CCT GTT TAC CAA AAA CAT 3’ tential gene bank for aquaculture. For stocking opera- Reverse: 5’ GGT ATG GGC CCG AAA GCT TA 3’ tions, knowledge of the genetic status of individual The PCR procedure applied to each sample in- stocks permits maintaining biodiversity resulting from volved 10 μl genomic DNA, 0.2 μM of each primer, 1 the geographic isolation of individual populations. In x PCR buffer [10 mM Tris-HCl (pH 8.3), 50 mM extreme cases, heterogeneous populations will be KCl], 1.5 mM MgCl2, 80 μg BSA, 0.1 mM dNTPs identified, and it will be recommended that they are mix, and 0.5 units Taq DNA-polymerase (Fermentas, excluded from further exploitation. Germany) in a total volume of 50 ìl. The PCRs were performed in a thermal cycler (Eppendorf, Germany) programmed for initial denaturation for 3 minutes at Materials and Methods 95°C followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 30 seconds, Tissue or fin clip samples of 114 whitefish individu- and extension at 72°C for 2.5 minutes. A final exten- als were collected from eight Polish lakes (Iñsko, sion at 72°C lasted for 10 minutes. The PCR Unauthenticated Download Date | 12/16/15 2:05 AM Genetic variability in European populations of Coregonus lavaretus (L.): an assessment based... 199 products obtained were digested with ten restriction Results enzymes (Fermentas, Germany). They were selected using Webcutter software 2.0 (available at All digested samples were easily distinguishable on http://rna.lundberg.gu.se/cutter2/) based on a refer- agarose gels. Compared to the previous coregonid ence mtDNA sequence for Coregonus lavaretus ob- study and applying the same ten restriction enzymes, tained from GenBank (accession number only one additional haplotype could be detected in AB034824). Unless indicated otherwise, the en- the ND-1 gene region. It was very similar to zymes were four-base cutters: XbaI (six-base cutter), haplotype H2, and it differed in the fragment pattern Eco47I (five-base cutter), HinfI (five-base cutter), of only one restriction enzyme (Eco47I: C instead of BsuRI, RsaI, AluI, MboI, HpaI, Hin6I, and TaqI. Di- A). This new haplotype HN (N – like new) was ob- gestion proceeded in reaction mixtures of 15 ìl total served in a single Lucerne sample (Table 1, and L. volume consisting of 10 ìl of the PCR product, 1.5 ìl Lucerne7 in Fig. 2). buffer adjusting the reaction conditions to the opti- H3 48 ì ì TRFL94 mum, 0.2 l of the restriction enzyme, and 3.3 l 60 TRFL50 sterile water. The resulting fragments were separated 61 TR233 H4 by electrophoresis on 2% agarose gel with the TBE 95 75 H5 buffer system, stained with ethidium bromide (EtBr), H6 and compared to peqGOLD 100 bp DNA-Ladder 60 TRFL48 L. Lucerne7 Plus (Peqlab Biotechnologie, Germany) size stan- 99 C. lavaretus reference 63 dards using BIO-1D Analysis Software for Electro- H1 39 H2 phoresis Images (Vilber Lourmat, France). The 57 L. Lucerne1 fragment patterns observed were marked with capi- H7 68 H8 tal letters starting from A, which was given to the ex- 100 C. albula,L.Narie,Poland pected pattern from the “virtual” digestion of the 54 C. albula,L.Stechlin,Germany H9 reference sequence. Letters from B on were used to 50 53 C. peled,L.Czarne,Poland mark the patterns deviating from expected pattern A. 0.002 94 C. peled,L.Czarne,Poland Subsequently, composite haplotypes were desig- Figure 2. Neighbor-joining tree based on Jukes-Cantor distances nated based on combinations of restriction fragments (upper branch group number one, lower branch group number resulting from the different restriction enzymes. two). Representative individuals of all observed com- Haplotype H2 was expressed with the highest posite haplotypes were sequenced on a CEQ 8000 frequency (68.42%); it was present in samples col- capillary sequencer (Beckman Coulter, USA). The lected from all the lakes studied (Table 2). The rarest preparation of internal primers, sequencing parame- haplotypes were H6, H7, and HN, for each of which ters, and the assemblage of the accumulated se- there was only one individual per haplotype (1.19%). quence fragments was performed according to As confirmed by the sequence analysis, haplotype Kohlmann et al. (2007). Sequences representing in- H1 was expressed in as few as two specimens only dividual haplotypes were also virtually digested to caught in the Lake Œremskie. In the sequence align- confirm the restriction sites obtained by the ten en- ment for 1929 nucleotides, 62 variable sites were de- zymes. In addition, to reconstruct phylogenetic rela- tected.