Isolation and characterization of eleven microsatellite loci of Pseudobagrus truncatus in the Yangtze River
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Isolation and characterization of eleven microsatellite loci of Pseudobagrus truncatus in the Yangtze River Huatang Deng • Xiaoyun Zeng • Dengqiang Wang Xinbin Duan • Daqing Chen
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Received: 3 December 2014 / Accepted: 23 December 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Eleven polymorphic microsatellite loci were characterized from a wild population of Pseudobagrus truncatus, an endemic catfish to China, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The number of alleles per locus ranged from 4 to 17, with the observed and expected heterozygosity ranging from 0.5087 to 1.000, and from 0.5937 to 0.9368, respectively. Cross-amplification was also tested in closely related species. These loci could be used for future assessment of genetic variation of P. truncates. Keywords Pseudobagrus truncatus Microsatellite Genetic marker Pseudobagrus truncatus, which distributes in the Yangtze River, is a small-sized benthonic catfish endemic to China (Ding 1994). This fish used to be one of the most popular species and has a great economic value (Zhou et al. 2014). Recently, however, wild population of P. truncatus declined dramatically and almost vanished in the main stream due to hydroelectric dam construction, over-fishing and water pollution. It is urgent and necessary to develop
Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0415-1) contains supplementary material, which is available to authorized users. H. Deng X. Zeng School of Life Science, Southwest University, Chongqing 400715, People’s Republic of China H. Deng D. Wang X. Duan D. Chen (&) Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Wuhan 430223, People’s Republic of China e-mail: [email protected]; [email protected]
genetic studies on the natural population aiming to conservation of the stock. These microsatellite markers which we reports here are suitable for quantifying genetic variability and population genetic structure, enabling to provide a more informed conservation strategy for this species. Genomic DNA was extracted from alcohol preserved fin tissue and digested with the restriction enzyme Mse I. A microsatellite DNA enriched library was constructed using the FIASCO method (Zane et al. 2002) using biotinylated (AC)15 or (CT)15 probes. DNA fragments containing microsatellite DNA were cloned into the pMD18-T vector (TaKaRa) and transferred into Escherichia coli DH5a competent cells. Positive clones were identified by PCR using M13 forward and reverse primers. 96 positive clones was sequenced successfully, 58 (60.4 %) of which containing repeat motifs. Microsatellite sequences were screened using the SSRHunter software (Li and Wan 2005). Thirty-five primer pairs were designed using Premier Prime 5.0 (Premier Biosoft International, Palo Alto, CA). Polymorphism at each locus was estimated using a wild population of 46 individuals. PCR amplifications were perform
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