Research
New microsatellite multiplex PCR sets for genetic studies of the sterlet sturgeon, Acipenser ruthenus
Klaus Kohlmann, Petra Kersten, Jörn Geßner, Dalia Onără, Elena Taflan, Radu Suciu
Abstract
Wild populations of the sterlet sturgeon, Acipenser ruthenus, are declining throughout their native ranges. In‑depth knowledge of their genetic diversity and structure is urgently needed to enable the identification of management units for conservation purposes. Moreover, genetic markers are required to establish appropriate breeding schemes for supportive stocking programs and to monitor genetic changes in farmed stocks. Therefore, six species‑specific, polymorphic microsatellite loci were isolatedand arranged into five multiplex PCR sets together with nine loci from other sturgeon species. The diversity of these 15 microsatellites was examined in 67 sterlet individuals (20 farmed in Germany and 47 wild‑caught in the Romanian part of the River Danube). The total number of alleles per locus ranged from 3 to 15 with an average of 7.20. The farmed sterlet sturgeon possessed 1 to 7 alleles per locus, with a mean of 3.13; the wild individuals were more variable, with 3 to 15 alleles per locus and a mean of 7.07. Observed heterozygosities ranged from 0 to 0.850 in the farmed individuals, and from 0.064 to 0.957 in the wild individuals. Indications of inbreeding were only found in the wild sterlet sturgeon (FIS=0.062). The genetic differentiation of the two sterlet groups was significant (FST=0.1186). The high sensitivity and discriminatory power of the 15 loci was indicated by the very low overall probability of identity for siblings (PIsib=5.099x10-5) and the high accuracy of self‑classification (66 out of the 67 individuals (98.51%) were correctly identified). Thus, these newly developed multiplex PCR sets are a valuable genetic tool for identifying management units for species conservation, sustainable fisheries and aquaculture.
References
| Altschul, S.F., W. Gish, W. Miller, E.W. Myers, D.J. Lipman. 1990. Basic Local Alignment Tool. Journal of Molecular Biology 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2 |
|||
| Barmintseva, A.E., N.S. Mugue. 2013. The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms. Russian Journal of Genetics 49: 950-961. https://doi.org/10.1134/S1022795413090032 |
|||
| Blankenship, H.L., K.M. Leber. 1995. A responsible approach to marine stock enhancement. American Fisheries Society Symposium 15: 167-175. | |||
| Cornuet, J.M., S. Piry, G. Luikart, A. Estoup, M. Solignac. 1999. New methods employing multilocus genotypes to select or exclude populations as origins of individuals. Genetics 153: 1989-2000. | |||
| Dudu, A., R. Suciu, M. Paraschiv, S. E. Georgescu, M. Costache, P. Berrebi. 2011. Nuclear markers of Danube sturgeons hybridization. International Journal of Molecular Sciences 12: 6796-6809. https://doi.org/10.3390/ijms12106796 |
|||
| Dudu, A., S. E. Georgescu, A. Burcea, I. Florescu, M. Costache. 2013. Microsatellites variation in sterlet sturgeon, Acipenser ruthenus from the lower Danube. Scientific Papers: Animal Science and Biotechnologies 46: 90-94. | |||
| Fopp-Bayat, D., G. Furgała Selezniow. 2010. Application of microsatellite DNA variation in Russian sturgeon (Acipenser gueldenstaedti) and sterlet (Acipenser ruthenus) cultured in a Polish fish farm. Polish Journal of Natural Sciences 25: 173-181. https://doi.org/10.2478/v10020-010-0014-x |
|||
| Gesner, J., J. Freyhof, M. Kottelat. 2010. Acipenser ruthenus. The IUCN Red List of Threatened Species 2010: e.T227A13039007.
http://dx.doi.org/10.2305/IUCN.UK.2010-1.RLTS.T227A13039007.en |
|||
| Goudet, J. 2002. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3.2). (http://www2.unil.ch/popgen/softwares/fstat.htm accessed August 22, 2008). | |||
| Havelka, M., T. Fujimoto, S. Hagihara, S. Adachi, K. Arai. 2017. Nuclear DNA markers for identification of beluga and sterlet sturgeons and their interspecific bester hybrid. Scientific Reports 7: 1694. https://doi.org/10.1038/s41598-017-01768-3 |
|||
| Henderson Arzapalo, A., T.L. King. 2002. Novel microsatellite markers for Atlantic sturgeon (Acipenser oxyrinchus) population delineation and broodstock management. Molecular Ecology Notes 2: 437-439. https://doi.org/10.1046/j.1471-8286.2002.00262.x |
|||
| Kaplinski, L., R. Andreson, T. Puurand, M. Remm. 2005. MultiPLX: automatic grouping and evaluation of PCR primers. Bioinformatics 21: 1701-1702. https://doi.org/10.1093/bioinformatics/bti219 |
|||
| Kohlmann, K., P. Kersten. 2006. Microsatellite loci in tench: isolation and variability in a test population. Aquaculture International 14: 3-7. https://doi.org/10.1007/s10499-005-9009-y |
|||
| Kohlmann, K., P. Kersten. 2008. Isolation and characterization of nine microsatellite loci from the pike perch, Sander lucioperca (Linnaeus, 1758). Molecular Ecology Resources 8: 1085-1087. https://doi.org/10.1111/j.1755-0998.2008.02166.x |
|||
| Larkin, M.A., G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, D.G. Higgins. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948. https://doi.org/10.1093/bioinformatics/btm404 |
|||
| Lorenzen, K., K.M. Leber, H.L. Blankenship. 2010. Responsible approach to marine stock enhancement: an update. Reviews in Fisheries Science 18: 189-210. https://doi.org/10.1080/10641262.2010.491564 |
|||
| Ludwig, A., N.M. Belfiore, C. Pitra, V. Svirsky, I. Jenneckens. 2001. Genome duplication events and functional reduction of ploidy levels in sturgeon (Acipenser, Huso and Scaphirhynchus). Genetics 158: 1203-1215. | |||
| Ludwig, A., S. Lippold, L. Debus, R. Reinartz. 2009. First evidence of hybridization between endangered sterlets (Acipenser ruthenus) and exotic Siberian sturgeons (Acipenser baerii) in the Danube River. Biological Invasions 11: 753-760. https://doi.org/10.1007/s10530-008-9289-z |
|||
| Malausa, T., A. Gilles, E. Meglécz, H. Blanquart, S. Duthoy, C. Costedoat, V. Dubut, N. Pech, P. Castagnone Sereno, C. Délye, N. Feau, P. Frey, P. Gauthier, T. Guillemaud, L. Hazard, V. Le Corre, B. Lung Escarmant, P. J. Malé, S. Ferreira, J. F. Martin. 2011. High throughput microsatellite isolation through 454 GS FLX Titanium pyrosequencing of enriched DNA libraries. Molecular Ecology Resources 11: 638-644. https://doi.org/10.1111/j.1755-0998.2011.02992.x |
|||
| May, B., C.C. Krueger, H.L. Kincaid. 1997. Genetic variation at microsatellite loci in sturgeon: primer sequence homology in Acipenser and Scaphirhynchus. Canadian Journal of Fisheries and Aquatic Sciences 54: 1542-1547. https://doi.org/10.1139/f97-061 |
|||
| McQuown, E.C., B.L. Sloss, R.J. Sheehan, J. Rodzen, G.J. Tranah, B. May. 2000. Microsatellite analysis of genetic variation in sturgeon: new primer sequences for Scaphirhynchus and Acipenser. Transactions of the American Fisheries Society 129: 1380-1388. https://doi.org/10.1577/1548-8659(2000)129<1380:MAOGVI>2.0.CO;2 |
|||
| Meglécz, E., C. Costedoat, V. Dubut, A. Gilles, T. Malausa, N. Pech, J. F. Martin. 2010. QDD: a user friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics 26: 403-404. https://doi.org/10.1093/bioinformatics/btp670 |
|||
| NCBI GenBank. https://www.ncbi.nlm.nih.gov/genbank/, accessed April 21, 2017. | |||
| Reinartz, R., S. Lippold, D. Lieckfeldt, A. Ludwig. 2011. Population genetic analyses of Acipenser ruthenus as a prerequisite for the conservation of the uppermost Danube population. Journal of Applied Ichthyology 27: 477-483. https://doi.org/10.1111/j.1439-0426.2011.01693.x |
|||
| Rousset, F. 2008. GENEPOP'007: a complete re implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources 8: 103-106. https://doi.org/10.1111/j.1471-8286.2007.01931.x |
|||
| Rozen, S., H. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods in Molecular Biology 132: 365-386. | |||
| Van Oosterhout, C., W.F. Hutchinson, D.P.M. Wills, P. Shipley. 2004. MICRO CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4: 535-538. https://doi.org/10.1111/j.1471-8286.2004.00684.x |
|||
| Waits, L.P., G. Luikart, P. Taberlet. 2001. Estimating the probability of identity among genotypes in natural populations: cautions and guidelines. Molecular Ecology 10: 249-256. https://doi.org/10.1046/j.1365-294X.2001.01185.x |
|||
| Welsh, A.B., M. Blumberg, B. May. 2003. Identification of microsatellite loci in lake sturgeon, Acipenser fulvescens, and their variability in green sturgeon, A. medirostris. Molecular Ecology Notes 3: 47-55. https://doi.org/10.1046/j.1471-8286.2003.00346.x |
|||
| Wilberg, M.J., B.P. Dreher. 2004. GENECAP: a program for analysis of multilocus genotype data for non invasive sampling and capture recapture population estimation. Molecular Ecology Notes 4: 783-785. https://doi.org/10.1111/j.1471-8286.2004.00797.x |
|||
| Zane, L., T. Patarnello, A. Ludwig, F. Fontana, L. Congiu. 2002. Isolation and characterization of microsatellites in the Adriatic sturgeon (Acipenser naccarii). Molecular Ecology Notes 2: 586-588. https://doi.org/10.1046/j.1471-8286.2002.00328.x |
|||