MI CROSATELLI TE LETTERS

Isolation and characterization of twenty polymorphic

microsatellites for Hemibarbus labeo (Cyprinidae)
Cuiyun Lu

Ying Gu

Longwu Geng

Lei Cheng

Chao Li

Guangxiang Tong

Haifeng Jiang

Shahid Mahboob

Wei Xu

Xiaowen Sun
Received: 21 July 2014 / Accepted: 19 August 2014
Springer Science+Business Media Dordrecht 2014
Abstract Hemibarbus labeo is a valuable freshwater sh
species distributed in the Heilongjiang River basin in
China. Wild resources have declined sharply in recent
years because of overshing and pollution. Herein, twenty
polymorphic microsatellites were isolated from an enriched
genomic library in H. labeo for genetic conservation. These
markers were characterized in 27 wild and 48 cultured
individuals. The number of alleles per locus ranged from
two to ten, with an average of 5.200. The observed heter-
ozygosity and the expected heterozygosity varied from
0.086 to 0.870 (mean = 0.538) and from 0.116 to 0.861
(mean = 0.5441), respectively. Three and two loci signif-
icantly deviated from HardWeinberg equilibrium in wild
and cultured individuals, respectively. Three pairwise
comparisons of six loci showed signicant genotypic
linkage disequilibrium in cultured individuals. These
polymorphic markers represent a powerful tool to study
population genetic diversity, genetic conservation and
breeding systems in H. labeo.
Keywords Hemibarbus labeo Microsatellite Genetic
conservation
Hemibarbus labeo is a small freshwater sh species dis-
tributed in eastern Asia, from the Heilongjiang River basin
to northern Vietnam, and in Japan and the Hainan and
Taiwan islands (http://www.shbase.org/summary/Hemi
barbus-labeo.html). This species is a valuable sh in
northeast of China because of its delicious taste. However,
the wild resources of H. labeo have declined sharply in
recent decades because of overshing and environmental
pollution. Fortunately, this species has been successfully
articially domesticated and bred for conservation and
sustainable usage (Xu et al. 2009). However, further
studies regarding conservation genetics and stock
enhancement of H. labeo are required.
Microsatellites are ideal tools for population genetics
because of their high polymorphism and co-dominant
inheritance. A previous study developed ten microsatellites
using PCR-based isolation of microsatellite arrays (PIMA)
in H. labeo (Lin et al. 2007). Here, we isolated twenty
polymorphic microsatellites from an enriched (CA)
12
genomic library and characterized them in wild and cul-
tured H. labeo.
Microsatellite sequences were isolated from an enriched
genomic library for (CA)
12
following the methods of Geng
et al. (2012). Briey, total DNA was extracted from the n
clip of an H. labeo individual and digested with Sau3AI
restriction endonuclease. DNA fragments (400900 bp)
were isolated, puried and ligated with short adaptors. The
ligated products were used as templates for a rst round of
polymerase chain reaction (PCR). PCR products were
hybridized with a biotin-labeled dinucleotide repeat (CA)
12
probe attached to magnetic beads. The second round of
Electronic supplementary material The online version of this
article (doi:10.1007/s12686-014-0299-0) contains supplementary
material, which is available to authorized users.
C. Lu Y. Gu L. Geng L. Cheng C. Li G. Tong
H. Jiang W. Xu (&) X. Sun (&)
National Local Joint Engineering Laboratory for Freshwater Fish
Breeding, Heilongjiang River Fisheries Research Institute,
Chinese Academy of Fishery Sciences, Harbin 150070, Peoples
Republic of China
e-mail: xwsc23@tom.com
X. Sun
e-mail: sunxw2002@163.com
S. Mahboob
Department of Zoology College of Science, King Saud
University, Riyadh 11451, Saudi Arabia
1 3
Conservation Genet Resour
DOI 10.1007/s12686-014-0299-0
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Conservation Genet Resour
1 3
PCR was conducted using enriched fragments as templates.
The products were ligated into vector pMD 18-T (Takara,
Bio Inc., Otsu, Shiga, Japan) and then transformed into
competent Escherichia coli DH5a. An additional hybrid-
ization was carried out to identify positive clones with a 5
0
-
[c-
32
P] ATP-labelled probe (CA)
12
.
Among 168 sequenced positive clones, 144 microsatellite
containing sequences were identied. Primer3 (version 0.4.0)
software (http://bioinfo.ut.ee/primer3-0.4.0) designed 85 pri-
mer pairs based on these sequences. The obtained markers
were characterized in 75 individuals (27 wild samples col-
lected from the Heilongjiang and Wusulijiang Rivers; 48
cultured individuals from the Hulan Aquaculture Experi-
mental Station, Harbin, China). An Applied Biosystems

GeneAmp

PCR System 9700 (Life Technologies Corpora-

tion, Carlsbad, CA, USA) performed the amplications. The
PCRreactions (15 lL) comprised100 ngDNA, 10 mMTris
Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl
2
, 200 lM each
dNTP, 0.2 mM primer and 1 U Taq DNA polymerase. The
PCR conditions comprised initial denaturation at 94 C for
3 min; followed by 27 cycles of 94 Cfor 30 s, the annealing
temperature (Table 1) for 30 s, 72 C for 30 s; and a nal
extension at 72 C for 5 min. Amplicons were subsequently
electrophoresed and sized on 8 % polyacrylamide gel and
visualized by silver staining. Gel-Pro Analyzer software
(Version 4.0) then determined the sizes of the amplicons by
comparison with DL1000 markers (Takara, Bio Inc.).
Twenty microsatellites displayed polymorphic bands in
75 H. labeo individuals. Popgene 32 (Yeh et al. 2000) ana-
lyzed the population genetics of these markers (Table 1).
The mean number of alleles (N
o
) and effective alleles (N
e
)
per locus were 5.200 and 3.017, respectively. The mean
observed heterozygosity (H
o
) was 0.538 and the mean
expected heterozygosity (H
e
) was 0.5441. GENEPOP ver-
sion 4.1 (Raymond and Rousset 1995) calculated the Hardy
Weinberg equilibriumand genotypic linkage disequilibrium
for all loci within the population. Three (HLJCH24,
HLJCH39, and HLJCH80) and two loci (HLJCH17 and
HLJCH44) deviated signicantly from HardyWeinberg
equilibrium after Bonferroni correction in wild and cultured
individuals, respectively (P\0.0025). The small sample
size and a non-randomly mating population are two probable
factors affecting this deviation. Additionally, three pairwise
comparisons among six loci (HLJCH02 and HLJCH14;
HLJCH44 and HLJCH45; HLJCH10 and HLJCH47)
showed signicant linkage disequilibrium in cultured indi-
viduals (P\0.0005) because of the limited number of
parental sh. These polymorphic microsatellites provide a
basis for population genetic diversity, genetic conservation,
and breeding system studies in H. labeo.
Acknowledgments This study was supported by the National
Infrastructure of Fishery Germplasm Resources of China.
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T
G
T
C
C
A
(
C
A
)
1
7
5
6
1
6
3

2
7
3
9
/
2
.
9
8
6
0
.
6
7
4
/
0
.
6
7
2
0
.
0
0
0
0
*
*
0
.
6
0
7
1
K
M
0
1
8
2
9
3
R
:
T
T
G
T
G
G
T
G
C
A
C
A
T
T
C
T
G
T
C
C
H
L
J
C
H
8
1
F
:
T
C
C
C
C
C
T
A
C
C
T
A
C
A
T
T
C
T
G
C
(
G
A
)
1
5
G
5
0
1
6
2

2
2
8
4
/
1
.
1
3
1
0
.
0
8
6
/
0
.
1
1
6
0
.
2
3
6
1
1
.
0
0
0
0
K
M
0
1
8
2
9
4
R
:
C
C
C
C
A
C
T
G
G
G
A
G
T
C
T
T
C
A
T
A
T
(
G
A
)
1
1
H
L
J
C
H
8
2
F
:
T
C
G
A
C
G
A
T
C
A
A
A
G
T
A
G
A
A
A
C
A
A
G
(
G
A
)
1
9
5
6
1
6
7

1
8
1
3
/
1
.
8
3
0
0
.
5
8
5
/
0
.
4
5
7
1
.
0
0
0
0
0
.
0
0
4
4
K
M
0
1
8
2
9
5
R
:
G
T
C
T
G
G
G
G
T
T
C
C
C
C
T
T
A
C
A
T
T
a
A
n
n
e
a
l
i
n
g
t
e
m
p
e
r
a
t
u
r
e
,
N
o
n
u
m
b
e
r
o
f
a
l
l
e
l
e
s
,
N
e
n
u
m
b
e
r
o
f
e
f
f
e
c
t
i
v
e
a
l
l
e
l
e
s
,
H
o
o
b
s
e
r
v
e
d
h
e
t
e
r
o
z
y
g
o
s
i
t
y
,
H
e
e
x
p
e
c
t
e
d
h
e
t
e
r
o
z
y
g
o
s
i
t
y
,
P
H
W
E
p
r
o
b
a
b
i
l
i
t
y
v
a
l
u
e
s
f
o
r
e
x
a
c
t
t
e
s
t
s
o
f
H
a
r
d
y

W
e
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n
b
e
r
g
e
q
u
i
l
i
b
r
i
u
m
*
P
\
0
.
0
0
2
5
,
*
*
P
\
0
.
0
0
0
5
Conservation Genet Resour
1 3
References
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characterization of twenty microsatellite markers for the endan-
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Raymond M, Rousset F (1995) GENEPOP (version 1.2): population
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Conservation Genet Resour
1 3