Conservation Genet Resour

DOI 10.1007/s12686-014-0194-8

MICROSATELLITE LETTERS

Isolation and characterization of fourteen polymorphic

microsatellite 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 freshwater 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 isolated 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
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. Yang
Freshwater Aquaculture Collaborative Innovation Center of
Hubei Province, Wuhan 430070, Peoples Republic of China

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 constructing 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. Seventyeight primer pairs were designed successfully based on
suitable flanking sequences. Thirty individuals of O. sinensis were collected from Liangzi Lake, a shallow accessorial 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 containing 1 9 Taq buffer (Takara), 0.5 lmol/L each of primer, 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, followed
by 35 cycles of denaturation at 95 C for 30 s, locus-specific annealing temperature (Table 1) for 30 s, 72 C for
45 s, and a final extension at 72 C for 10 min. Amplified
products were electrophorized in 8 % non-denaturing

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KJ546088
KJ546089
KJ546090
KJ546091
KJ546092
KJ546093

(CA)12

(AAT)5

(GTCT)17

(GATG)5

(TCCA)5

(CA)7(CA)5(GACA)24(CA)5

(GT)6(TG)53(TG)7

(ACAG)30(GAC)5

(GT)33

(GT)5(TG)13(GT)6(TG)12

(GT)5

(TG)10

Os3

Os4

Os5

Os6

Os7

Os8

Os9

Os10

Os11

Os12

Os13

Os14

F: AAGCATACAGAAGTCGTAGT

F: ACGGGACAAAGCGGTAGA

F: AAGACTGAATGATGGTAGG
R: AACAAGCAACACAGAACAC

R: GTCCAACCTGGGAACACTA

F: GCCTAATGCGTTTGATGA

R: ATAACCTGCTACCCCCAT

F: ACAGTCCCTCTGAAAACC

R: AGTCATTTCCCAGCCTCT

F: GTTGCCACTGCCTTTGTT

R: CTGAGTAAGGGGTCTGTC

F: GTCTGAAAGGATGTGGTT

R: AACGGACAAAGCGGTAGA

F: ACGGGTCAGAGAGGGAGT

R: ACGGGTCAGAGAGGGAGT

53

53

55

55

55

54

60

57

R: ACTATGTCTCTCTGCTGGC

57

F: ACCTGTTTCGGATGCCTC

54

55

54

54

R: TGCGTATGCTGTTCTTGGA
F: ATGTGCGTATGCTGTTCT

R: TGACTGAGGGTCGCTTTC

F: TGATGTGAGTGATGTGGGA

R: CAGACCCCAACAACATAGC

F: GAGAATCAGCACCTCCAAG

R: TAGCAGACAACACTGTAACT

F: ACTCAGGACTCATCATCAAC

R: AGTTACTCAGGACTCATCAG

52

Ta (C)

14

14

Na

280349

316330

260290

176219

286336

172236

158176

196200

204238

260298

190242

240284

166186

152184

Size (bp)

* Significant deviation from HardyWeinberg equilibrium (HWE) (P \ 0.05)

Ta annealing temperature (C), Na number of alleles, HO observed heterozygosity, HE expected heterozygosity, PIC polymorphic information content

KJ546095

KJ546094

KJ546087

KJ546086

KJ546085

KJ546084

KJ546083

(GT)7(CTGT)16

Os2

F: ACTCATCGTCACTCTTCATT
R: GTAAGGTAAGCATACAGAAGTC

KJ546082

(CA)12

Os1

Primer sequence (50 30 )

Repeat

Locus

Accession no.

Table 1 Characterization of 14 polymorphic microsatellite loci of Odontobutis sinensis

0.533

0.633

0.067

0.367

0.567

0.533

0.433

0.133

0.467

0.333

0.433

0.367

0.500

0.700

HO

0.558

0.779

0.631

0.516

0.628

0.887

0.345

0.500

0.638

0.291

0.445

0.866

0.557

0.689

HE

0.460

0.748*

0.566*

0.462

0.552

0.877*

0.282

0.375*

0.584*

0.271

0.402

0.854*

0.508

0.643

PIC

Conservation Genet Resour

Conservation Genet Resour

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 correction 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 heterozygosities 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 Bonferroni correction. These newly isolated polymorphic loci will
be useful for population genetics analysis and effective
conservation of O.sinensis and related species.

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Acknowledgments This work was supported by Special Fund for

Agro-scientific Research in the Public Interest (Grant No. 201403012)
and Project of Liangzi Lake Wetland Restoration.

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