MICROSATELLITE LETTERS

Isolation and characterization of fourteen polymorphicmicrosatellite loci for Odontobutis sinensis

Ping Qiu Xuefen Yang Kai-Jian Wei

Zhihong Ma Ruibin Yang

Received: 24 March 2014 / Accepted: 3 April 2014

Springer Science+Business Media Dordrecht 2014

Abstract Odontobutis sinensis is an indigenous fresh-

water fish in China. The wild population size of this species

has declined sharply in inland waters in recent years. For

the purpose of the conservation of natural resources of O.

sinensis, 14 polymorphic microsatellite loci were isolated

and characterized in 30 individuals collected from Liangzi

Lake in Hubei Province. The number of alleles per locus

ranged from 2 to 14 with an average of 5.07. The expected

and observed heterozygosities ranged from 0.291 to 0.887

and from 0.067 to 0.700, respectively. These newly iso-

lated loci are currently being used for population genetic

diversity analysis and will be valuable for the conservation

of O. sinensis.

Keywords Odontobutis sinensis Microsatellite loci Polymorphism

Odontobutis sinensis, an indigenous freshwater fish species

in China, is mainly distributed in shallow lakes along the

middle reaches of the Yangtze River (Wu et al. 2002).

Among the community of indigenous freshwater fishes of

China, it is peculiar that the embryonic development of O.

sinensis is very long and most of the organ developments is

basically completed before its larva get out of the egg

membranes. In recent years, the wild resources of this

species have declined sharply in inland waters due to

overfishing and water environment deterioration. In order

to conserve natural resources of O. sinensis, it is essential

to understand its genetic diversity and population genetic

structure. However, there is limited genetic information

about this species except for little studies on its karyotype

analysis, phylogeny and morphological characteristics. In

this study, fourteen polymorphic microsatellite loci were

isolated in O. sinensis in order to further estimate its

genetic diversity and population genetic structure.

The genomic DNA was extracted from fin tissue

digested by a traditional proteinase-K. Microsatellite loci

were isolated using the fast isolation by AFLP of sequences

containing repeats (FIASCO) protocol (Zane et al. 2002)

with magnetic beads enrichment. The methods of con-

structing microsatellite enriched libraries and screening

positive clones were referred to Li et al. (2009). In total,

145 clones possibly with microsatellite fragments were

sent for sequencing. Eighty-seven of the sequenced clones

were detected to contain simple sequence repeats. Seventy-

eight primer pairs were designed successfully based on

suitable flanking sequences. Thirty individuals of O. sin-

ensis were collected from Liangzi Lake, a shallow acces-

sorial lake of the Yangtze River, and were used for

polymorphic test of isolated microsatellite loci.

Fourteen out of the 78 primer pairs were tested to be

polymorphic and produced expected PCR products. PCR

amplifications were conducted in a 10 lL volume con-taining 1 9 Taq buffer (Takara), 0.5 lmol/L each of pri-mer, 200 lmol/L dNTPs, 0.5 U Taq DNA polymerase(Takara) and 50 ng genomic DNA under the following

program: a pre-denaturation at 95 C for 5 min, followedby 35 cycles of denaturation at 95 C for 30 s, locus-spe-cific annealing temperature (Table 1) for 30 s, 72 C for45 s, and a final extension at 72 C for 10 min. Amplifiedproducts were electrophorized in 8 % non-denaturing

P. Qiu X. Yang K.-J. Wei Z. Ma R. Yang (&)Key Laboratory of Freshwater Animal Breeding, Ministry of

Agriculture, College of Fisheries, Huazhong Agricultural

University, Wuhan 430070, Peoples Republic of China

e-mail: rbyang@mail.hzau.edu.cn

X. Yang K.-J. Wei R. YangFreshwater Aquaculture Collaborative Innovation Center of

Hubei Province, Wuhan 430070, Peoples Republic of China

123

Conservation Genet Resour

DOI 10.1007/s12686-014-0194-8

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polyacrylamide gel and visualized by silver staining. A

pUC18/Msp I marker (Tiangen) was used as the standard to

identify the sizes of alleles.

POPGENE 1.32 (Yeh et al. 1997) and CERVUS 3.0

(Kalinowski et al. 2007) were used to calculate number of

alleles, observed heterozygosity, expected heterozygosity,

polymorphic information content. Deviation from Hardy

Weinberg equilibrium (HWE) was tested at each locus

using POPGENE 1.32 and a sequential Bonferroni cor-

rection for multiple comparisons was applied to all loci

(Rice 1989). The number of alleles ranged from 2 to 14

with an average of 5.07. The expected and observed het-

erozygosities ranged from 0.291 to 0.887 and from 0.067 to

0.700, respectively. The polymorphic information content

ranged from 0.271 to 0.877. Six of the 14 loci showed

significant deviations (P \ 0.05) from HWE after Bonfer-roni correction. These newly isolated polymorphic loci will

be useful for population genetics analysis and effective

conservation of O.sinensis and related species.

Acknowledgments This work was supported by Special Fund forAgro-scientific Research in the Public Interest (Grant No. 201403012)

and Project of Liangzi Lake Wetland Restoration.

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Isolation and characterization of fourteen polymorphic microsatellite loci for Odontobutis sinensisAbstractAcknowledgmentsReferences