MICROSATELLITE LETTERS

Isolation and characterization of 14 microsatellite loci for Chinesecatfish Silurus asotus

Mingsong Xiao • Fangying Bao

Received: 11 August 2013 / Accepted: 17 September 2013 / Published online: 25 September 2013

� 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 Bon-

ferroni correction. These microsatellite loci will be useful

for revealing population structure, and conservation genet-

ics 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 reser-

voirs, lakes and rivers of China. Due to overfishing, envi-

ronmental 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

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-enri-

ched DNA fragments were amplified by PCR, and then

ligated into pGEM-T Easy vectors (Promega) and trans-

formed 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. Thirty-six positive

clones were screened and sequenced. Twenty primer sets

were designed through Primer 5.0 software (Clarke and

Gorley 2001) and synthesized. Instead of a 50 dye-labelled

primer, a M13F (-29) sequence was added to the 50 end of the

forward primer or the reverse primer. To produce labeled

DNA fragments, labelled M13F was added to the reaction.

PCR amplifications were conducted in 25 lL volumes

containing 100 ng template DNA, 12.5 lL Ex Taq premix

buffer (TaKaRa), 5 pmol of each primer, and 0.5 pmol of

fluorescently labelled M13 primer [either IRD700 or

IRD800 (LI-COR)]. The conditions for amplification were

5 min at 95 �C followed by 30 cycles of 30 s at 95 �C, 30 s

at the annealing temperature (Table 1) and 30 s at 72 �C

M. Xiao (&) � F. Bao

College of Life Sciences, Anhui Science and Technology

University, Fengyang 233100, China

e-mail: xiaomingsong2004@126.com

123

Conservation Genet Resour (2014) 6:163–165

DOI 10.1007/s12686-013-0035-1

with a final extension time of 10 min at 72 �C. PCR products

were separated on denaturing 6.5 % polyacrylamide gels

using a LI-COR 4300 automated DNA sequencer and ana-

lysed using LI-COR SAGAGT software.

The primers were tested for polymorphism on 30 indi-

viduals collected from the Huaihe River. Fourteen of 20

loci designed for S. asotus were polymorphic, while the

other six primer pairs amplified monomorphic loci or failed

to get a consistent amplification. The number of alleles at

each polymorphic locus, their size range, and observed and

expected heterozygosities was calculated using CERVUS

2.0 software (Marshall et al. 1998) (Table 1). The number

of alleles per locus ranged from nine to fifteen. Observed

and expected heterozygosities ranged from 0.593 to 0.931

and from 0.833 to 0.926, respectively. Deviation from

Hardy–Weinberg equilibrium and linkage disequilibrium at

each locus was calculated using Genepop 3.4 (Raymond

and Rousset, 1995). Four loci deviated from HWE in the

sampled population after Bonferroni correction (adjusted

P value = 0.0036), and the remaining 10 loci were in

HWE (Table 1). These deviations from expectations may

be due to insufficient sample size, the occurrence of null

alleles, or sampling of individuals from multiple distinct

populations since they were collected throughout a very

large river system. There was no evidence for linkage

disequilibrium (P [ 0.05) between all pairs of loci. The

allelic frequencies were less than 10 % for each locus using

Genepop 3.4. The microsatellite loci described here are

Table 1 Levels of variability at 14 polymorphic microsatellite loci in S. asotus

Locus Repeat motif Primer sequences (50–30) Size

range

(bp)

Ta

(�C)

n NA HO HE P values

(HWE)

F

(%)

GenBank

accession

no.

SS17 (AG)26 F: CTGTTTACAAGTTGGTGGTG

(tailed)

R: CAGGTAGCAGGAATGA

156–174 53 26 9 0.769 0.867 0.0066 9.98 JQ624588

SS31 (TG)8 F: CAGCAGCAGCGGAGAATC (tailed)

R: CGTCAAGGGAAAGTCGTA

141–157 52 28 9 0.821 0.836 0.2025 9.87 JQ624589

SS57 (AG)24 F: CAGTTCAGTCAGGTAGGAGGTT

R: GAGTTTGTGGAGGCGTG(tailed)

201–229 52 29 14 0.862 0.926 0.00007* 8.80 JQ624590

SS59 (CT)19 F: GACAGAACATAAGCGAACAC

R: AATCCATCCATCATAGCG (tailed)

249–265 54 29 9 0.793 0.849 0.0105 9.86 JQ624591

SS94 (CT)32(GA)7 F: GCTTCGGATCAGTAAAC (tailed)

R: CAGGTGGGACAAACAAT

247–279 52 30 15 0.900 0.901 0.0336 7.10 JQ624592

SS123 (TC)33 F: ACTGCTCTGGGAATGGTT(tailed)

R: TCACTCTGGCACTCGTTT

237–255 53 30 9 0.800 0.833 0.1002 9.42 JQ624593

SS127 (GA)31(TG)28 F: AAGTGTTGGGAAGTCTGA

R: CTCTTTGGGCTTGTGCTG (tailed)

176–198 52 28 12 0.857 0.905 0.0001* 8.22 JQ624594

SS172 (AG)10(GA)12 F: CAGAGGGTGGTGACGATG (tailed)

R: CAAGGGCTAGAGGGAATA

301–321 53 28 11 0.786 0.901 0.0020* 9.06 JQ624595

SS190 (CT)19 F: AGACAGAACATAAGCGAAC

(tailed)

R: AATCCATCCATCATAGCG

251–275 54 29 13 0.826 0.900 0.0058 7.64 JQ624596

SS198 (CT)27(CT)5 F: ATGTGGGTAGATGTTGTGC (tailed)

R: CTGAGTGGATGGAGTGGG

231–249 53 29 10 0.593 0.900 0.0052 9.94 JQ624597

SS209 (TG)15 F: GAAATCAGCCAATCAGAGG

R: CAGCCGAGTGTAAACAGC (tailed)

244–268 52 29 13 0.897 0.878 0.0412 8.31 JQ624598

SS272 (TG)16(TG)12 F: AGTTCGTCAGACACCAGGAT

R: AAACAAAGGGAAGCAGG (tailed)

224–250 55 29 11 0.931 0.901 0.0127 9.07 JQ624599

SSW1 (CA)32 F: CATAGTGGAGGAGGTGGC (tailed)

R: TCTGGTGTTTCTGGCATT

145–167 53 30 10 0.733 0.749 0.0307 9.96 JQ624600

SSW3 (TG)18 F: GGCTATCATTAGGAAGA

R: AGATTCGGCTATCATTAGG (tailed)

121–143 52 29 12 0.724 0.892 0. 0004* 9.06 JQ624601

Ta annealing temperature, n sample sizes, NA number of alleles, HO observed heterozygosity, HE expected heterozygosity, F null allelic

frequencies for each microsatellite loci. ‘tailed’ indicates M13F(-29) which is added to the 50-end of the primer

* Significant departure (P = 0.0036) from Hardy–Weinberg equilibrium after correction for multiple tests

164 Conservation Genet Resour (2014) 6:163–165

123

expected to be useful for further studies of the conservation

and fishery management for S. asotus.

Acknowledgments This research was supported by the Program for

the major project of the Natural Science Foundation of the Anhui

Higher Education Insitutions of China (KJ2011Z069).

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