Isolation and characterization of 14 microsatellite loci for Chinese catfish Silurus asotus
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MICROSATELLITE LETTERS
Isolation and characterization of 14 microsatellite loci for Chinese catfish Silurus asotus Mingsong Xiao • Fangying Bao
Received: 11 August 2013 / Accepted: 17 September 2013 / Published online: 25 September 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Silurus asotus is a high commercially valuable aquaculture fish in some regions of China. In this survey, the first set of 14 polymorphic microsatellite loci for S. asotus was developed and characterized. The number of alleles per locus ranged from 9 to 15 and the observed and expected heterozygosities ranged from 0.593 to 0.931 and from 0.833 to 0.926, respectively. Four loci were found deviated from HWE in the sampled population after Bonferroni correction. These microsatellite loci will be useful for revealing population structure, and conservation genetics of S. asotus. Keywords Silurus asotus Microsatellite Enrichment by magnetic beads
The Chinese catfish Silurus asotus is a high commercially valuable aquaculture fish in some regions of China. It used to be widely distributed throughout the freshwaters reservoirs, lakes and rivers of China. Due to overfishing, environmental pollution and other human disturbances in recent decades, this species has decreased rapidly and has almost disappeared in many river systems. Knowledge of the population genetic structure is important for management and sustainable utilization of this economic but threatened species. To data, only little information on molecular population genetics is available at present. Attempts to characterize population structure of S. asotus using RAPD and mitochondrial DNA Cytochrome b Gene sequence data have been hampered by the low variability of these genetic M. Xiao (&) F. Bao College of Life Sciences, Anhui Science and Technology University, Fengyang 233100, China e-mail: [email protected]
markers (Li et al. 2001; Wang et al. 2008). However, no S. asotus microsatellite loci have yet been published. Here, we cloned a suite of fourteen microsatellite markers from S. asotus. These microsatellite primers should be useful in a wide range of population and evolutionary studies of this species. Thirty individuals were collected from the upstream to downstream of the Huaihe River. Genomic DNA was extracted from muscle tissues using the DNeasy Tissue Kit (QIAGEN). DNA samples were pooled and digested with Sau3AI restriction enzyme. Size-selected fragments (300–800 bp) were ligated to Sau3AI adaptors (Refseth et al. 1997). The ligated fragments were hybridized with a 50 biotinylated probe (CA)15 at room temperature for 30 min and then captured by streptavidin-coated magnetic beads (Promega). After washing, the bound enriched single DNA fragments were eluted from the beads. Microsatellite-enriched DNA fragments were amplified by PCR, and then ligated into pGEM-T Easy vectors (Promega) and transformed into JM109 competent cells. Transformed cells grew at 37 °C for 16 h on LB agar plate containing ampicillin, X-gal and IPTG for blue/white selection. Th
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