Ñèáèðñêèé ýêîëîãè÷åñêèé æóðíàë, 2 (2016) 184193 ÓÄÊ 597.2/.5 DOI 10.15372/SEJ20160202 Evaluation of some microsatellite markers variability in the study of genetic structure of vendace (Coregonus albula (L.)) populations from Latvian Lakes N. KUTE, J. OREHA Institute of Life Sciences and Technology Daugavpils University LV-5400, Daugavpils, Parades str., 1A E-mail: nà[email protected], jelenà[email protected] Ñòàòüÿ ïîñòóïèëà 21.05.2015 Ïðèíÿòà ê ïå÷àòè 16.10.2015 ABSTRACT Vendace (Coregonus albula (L.)) is a very plastic species of freshwater whitefish which is widespread in Europe. But in Latvia this species is included into the list of specially protected fish species with limited use. We examined cross-species amplification of 14 microsatellite loci (Cocl-Lav22, Cocl-Lav23, Cisco-59, Cisco-106, Cisco-90, Cisco-126, Cisco-157, Cisco-179, Cisco-181, Cisco-183, Cisco-200, BWF1, BWF2, and C4-183) which were successfully used for genetic studies, monitoring, protection and management of different Coregonus species. Five microsatellite markers (BWF1, Cisco-90, Cisco-126, Cisco-157, Cisco-200) had a good representation in populations of vendace from five Latvian lakes (Sventes, Raznas, Nirzas, Dridzas, Stir- nu) and were used for genetic analysis of these populations. It was shown that these 5 microsatellite loci were polymorphic and could differ among the investigated populations. The mean number of alleles per locus (from 4.4 to 6.2), the observed (from 0.53 to 0.70) and expected (from 0.49 to 0.70) heterozygosity, population structure (FST and RST) and gene flow (Nm) were also analysed in Latvian vendace populations. Key words: Coregonus albula, microsatellites, Latvian lakes, polymorphism, heterozygosity, differen- tiation. Because of their widespread distribution, 20th century, Coregonus albula was artificially commercial importance and high phenotypic introduced from lakes Peipus and Ladoga to diversity throughout the northern hemisphere, more than 30 Latvian lakes. The vendace was coregonids have been the subject of intensive detected in 30 Latvian lakes in the 1930s. In research efforts for more than a century [Ber- the 30s the industrial catch of vendace reached nàtchez, 2004]. The vendace (Coregonus albu- 13 tons per lake. At the present moment this la) is a fish widely spread in Holarctic waters. species can be found only in some Latvian lakes It has reached North-western Europe after gla- and it is included in the list of specially pro- ciations. The vendace is a polymorphic species tected species in Latvia (Regulation No 396 of of circumpolar distribution composed of seve- the Cabinet of Ministers of the Republic of ral intraspecific forms. At the beginning of the Latvia, 14.11.2000). The lack of basic data on © kute N., Oreha J., 2016 184 biology and population status of the local ven- shown in Table 1. The location of lakes can be dace population hinders sustainable utilization seen in Fig. 1. Sample tissues were stored at of this fish and efforts to manage its repro- 20 C. DNA was extracted according to the duction in Latvian lakes. salt extraction method of S. M. Aljanabi, I. Mar- Microsatellites are more revealing over tinez [1997]. shorter geographical distances, where a few Microsatellite amplification was performed cases of panmixia [Dannewitz et al., 2005] and using the ABI 9700 biocycler. PCR was perfor- numerous cases of isolation by distance pat- med in 12 kl. PCR mixture components were: terns [OReilly et al., 2004], clinal variation a DNA sample, 10 Taq Buffer with KCl, [Nielsen et al., 2004], fragmentation [Lemaire 1.5 mM MgCl2, 2mM dNTP mix, 0.06 U/l Taq et al., 2005], and hybridization [Gum et al., DNA polymerase, 0.4 mol/l of each primers 2005] have been identified. Microsatellite geno- of 14 microsatellite loci (Cocl-Lav22, Cocl-Lav23, types are particularly useful for detecting struc- Cisco-59, Cisco-106, Cisco-90, Cisco-126, Cisco-157, ture in closely related populations, regardless Cisco-179, Cisco-181, Cisco-183, Cisco-200, BWF1, of whether they are in evolutionary equilibri- BWF2, and C4-157). PCR were performed us- um or not. But microsatellite markers that are ing the thermal cycling programme, following used to study vendace population are not de- an initial denaturation at 94 C for 5 min, 25 cyc- veloped well enough. Therefore the aim of this les were run with 94 C for 30 s, 56 C for work was to evaluate the variability of some 30 s, and 72 C for 60 s followed by 7-min ex- microsatellite markers and use them to study tension at 72 C and cooling at 4 C. PCR pro- the genetic structure of vendace populations ducts were fractioned by analytic electrophore- in Latvian lakes. sis in polyacrylamide gel (PAAG) according to the general recommendations [Paulauskas, Tu- belyte-Kirdiene, 2002], using TBE (0.045M Tris, MATERIALS AND METHODS 0,001M EDTA, 0,045M H3BO3, pH 8.38,4) The fish samples were collected in Latvia buffer in a vertical electrophoresis device. Ampli- in 2007, with the help of specialists of the fied microsatellites were being electrophoreti- Latvian Fish Resources Agency, from sampling caly separated for one hour using the follow- 1 1 catches according to Latvian Fish Resources ing mode: 10 V cm 20 min and 1617 V cm . Agency monitoring plan from seven Latvian The DNA marker (pUC19 DNA/Msp l (Hpa II) lakes: Sventes, Raznàs, Nirzas, Dridzas, Alu#ks- marker (3450 bp), (MBI Fermentas, Vil- nes and Eezers. The lake characteristics are nius, Lithuania)) was used. PCR products were T a b l e 1 The characteristics of Latvian lakes in which the vendace was collected Lake Location Area, ha Mean depth, m Max depth, m Ra#znàs 5619 N 5760 7 17 2727 E Nirzas 5623 N 550 8 21 2754 E Sventes 5551 N 740 8 38 2621 E Dridzis 5558 N 750 13 65 2718 E Stirnu 5633 N 150 8 26 2601 E Alu#ksnes 5727N 1540 7 15 275 E Eezers 5610 N 990 6 21 2735 E 185 Fig. 1. Location of lakes in Latvia, in which the vendace was collected visualized in UV light (302 nm) with EtBr was used to identify the null allele [Oosterhout (5 kg/ml), using the UVP visualization sys- et al., 2004]. The MICRO-CHECKER program tem. The size of microsatellites was determined helped to identify genotyping errors due to non- by comparing them to the marker using Vi- amplified alleles (null alleles), short allele domi- sionWorksLS software (Ultra-Violet Products nànce (large allele dropout) and scoring of stut- Ltd, UK). Amplification was repeated three ter peaks. It was also used to detect typographic times with each primer, including a positive errors. and negative control. The six microsatellite primers (Cisco-90, Cis- co-106, Cisco-126, Cisco-157, Cisco-200 and RESULTS AND DISCUSSION BWF1), which gave repeatable and informa- tive responds in testing, were selected for ana- Microsatellites are successfully used for ge- lysis. For genotyping the PCR products, which netic studies of different Coregonus species, were obtained with fluorescently-marked prim- as well as for monitoring, protection and mana- ers TMR, HEX, FAM, were subjected on ABI gement of this species. But microsatellite mar- 310 automated sequencer using Genescan LIZ kers that are used to study vendace popula- 500 (Applied Biosystem) as international size tion are not developed well enough. Since the standard. Number of alleles in a locus, fre- development of specific microsatellites primers quency, private alleles in each population, ob- requires time and material investments, it may served and expected heterozygosity level in be used to study the primers designed for re- polymorph loci were measured, their differ- lated species. For example, Rogers et al. [2004] ences and significance with criteria were cal- identified 31 microsatellite loci for the Canadi- culated using POPGENE 1.32 and GeneAlex an whitefish (Coregonus clupeaformiss, Mit- 6.41 software. The MICRO-CHECKER program chill). The authors tested the developed mar- 186 kers for six related fish species (Prosopium coulteri, Coregonus lavaretus, Oncorhynchus my- kiss, Salvelinus alpinus, C. artedi è Salmo sa- -value lar), which showed the applicability of these p markers for these species. Many researchers T a b l e 2 [e. g. Patton et al., 1997; Turgeon et al., 1999] exp H widely used the microsatellites markers de- signed for Coregonus clupeaformiss and C. nà- Heterozygosity sus in the study of population genetic of dif- obs ferent species of whitefish. The cross-species H amplification of microsatellite loci on Coregonus albula was shown [Huuskonen et al., 2004; Schulz et al., 2006; Praebel et al., 2013]. We examined the amplification of 14 microsatellite loci (Cocl-Lav22, Cocl-Lav23, Cisco-59, Cisco-106, Cisco-90, Cisco-126, Cisco-157, Cisco-179, Cisco- 181, Cisco-183, Cisco-200, BWF1, BWF2, C4-157) which were developed by J. Turgeon conditions PCR et al. [1999] and J. C. Patton et al. [1997]. These C) number of cycles markers were successfully used for genetic stu- ( a Microsatellite markers: description and amplification conditions T dies of different Coregonus species (e. g. C. nà- sus, C. artedi C. clupeaformis, C. hoyi and others) [Huuskonen et al., 2002, Schulz et al., 2006]. Five microsatellite markers (BWF1, Cis- HEX 53HEX 53 27 27 0.61 0.70 0.89 ns 0.95 ns co-90, Cisco-126, Cisco-157 and Cisco-200) had TMR 55TMR 53 30FAM 55 27 0.65 0.89 30 0.43 *** 0.51 0.66 * 0.76 *** a good representation in populations of ven- 3 dace from the following Latvian lakes: Sventes, 8 Ra#znàs, Nirzas, Drizis, Stirnu, Alu#ksnes, Ee- (GT) 25 17 45 zers. General characterization of these five mi- 84 N ATAT(AC) crosatellites primer pairs is described in Tab- 10 10 le 2.