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TECHNICAL NOTE
A first set of polymorphic microsatellite loci isolatedfrom Rhinogobio cylindricus
Hong Yan Liu • Fei Xiong • Xin Bin Duan •
Da Qing Chen • Shao Ping Liu • Fan Rrong Zhang •
Dong Yang • Lai Ning Yu
Received: 11 August 2011 / Accepted: 20 September 2011 / Published online: 1 October 2011
� Springer Science+Business Media B.V. 2011
Abstract Rhinogobio cylindricus is an endemic fresh-
water fish species with relevant commercial importance in
the Yangtze River, China. Nineteen polymorphic micro-
satellite loci were developed by fast isolation by AFLP of
sequences containing repeats (FIASCO) protocol. The
number of alleles, observed and expected heterozygosity
ranged from 2 to 6, from 0.3333 to 1.000 and from 0.2943
to 0.7695, respectively. Ten loci were highly informative,
nine were moderately informative. Six loci significantly
deviated from Hardy–Weinberg equilibrium after Bonferroni
correction. Two groups of couple loci exhibited significant
linkage disequilibrium. These polymorphic microsatellite
loci will provide useful genetic tools for population study
and conservation genetics of R. cylindricus.
Keywords Rhinogobio cylindricus � Microsatellite �Genetic conservation
Rhinogobio cylindricus, a medium-sized freshwater fish
species endemic to China, distribute in the main stream of
the Yangtze River and its tributaries. The fish lives in the
bottom of the river with rapid current and spawns pelagic
eggs (Ding 1994). Historically, R. cylindricus is one of the
most popular species with relevant commercial importance
in the Yangtze River region, however, now the wild
resource was severely damaged owing to over-fishing,
water pollution and the construction of large hydroelectric
projects (Ma and He 2004). Especially, the construction of
Three Gorges Dam has greatly changed the habitat con-
dition and reduced spawning grounds of R. cylindricus,
which led to a marked population decline of this species.
There were not yet many studies conducted on this fish
species until now. For conserving and recovering the
species through appropriate measures, genetic studies on
the population are necessary. Now, there is no microsat-
ellite marker study avialable on R. cylindricus. In this
study, we report the development of 19 polymorphic
microsatellite loci, which will provide powerful tools for
studying population genetics of this species.
Total DNA for constructing a microsatellite-enriched
library was extracted from muscle tissues of a pool of four
R. cylindricus individuals, using a traditional proteinase-K
digestion and phenol–chloroform protocol with slight
modifications (Liu et al. 2011). Repeat enriched genomic
library was constructed using the fast isolation by AFLP
of sequences containing repeats (FIASCO) protocol (Zane
et al. 2002). In brief, the whole genomic DNA was
digested with MseI restriction enzyme and ligated to the
adaptors (MseIA: 50-TAC TCA GGA CTC AT-30 and
MseIB: 50-GAC GAT GAG TCC TGA G-30). The micro-
satellites were enriched with the Biotin-labelled probes
containing the repeat motif (AC)12, streptavidin coated
magnetic beads. DNA with microsatellites captured by
magnetic beads was eluted in suitable sterilized distilled
water. These microsatellite-enriched DNA fragments were
PCR amplified using corresponding primers, cloned into
pGEM-T easy vector (Promega corporation), and then
transformed into Escherichia coli competent cell. Positive
clones verified through PCR were sequenced on an ABI
3730 automatic sequencer. Microsatellite sequences were
H. Y. Liu � F. Xiong � F. R. Zhang � D. Yang � L. N. Yu
College of Life Science, Jianghan University,
Wuhan 430056, China
e-mail: [email protected]
X. B. Duan (&) � D. Q. Chen � S. P. Liu
Yangtze River Fisheries Research Institute,
Chinese Academy of Fishery Sciences, Wuhan 430223, China
e-mail: [email protected]
123
Conservation Genet Resour (2012) 4:307–310
DOI 10.1007/s12686-011-9531-3
Ta
ble
1N
inet
een
po
lym
orp
hic
mic
rosa
tell
ite
loci
inR
hin
og
ob
iocy
lin
dri
cus
Lo
cus
Acc
essi
on
no
.R
epea
tm
oti
fP
rim
er(50 –
30 )
AS
ize
ran
ge
(bp
)H
OH
EP
PIC
RC
11
JN4
13
76
0(A
C)9
F:
AT
GG
CT
GC
TG
CC
TC
TG
AC
R:
TC
CA
GG
GA
GC
TT
TT
AG
AC
CA
21
59
–1
65
0.9
58
30
.50
98
0.0
00
00
.37
5
RC
12
JN4
13
76
1(T
G)1
7F
:T
AT
TG
GC
AA
TT
GG
CG
TG
TA
A
R:
CT
TT
CC
GA
GC
TT
GT
GG
AG
AC
61
74
–1
86
1.0
00
00
.74
29
0.0
30
10
.68
5
RC
18
JN4
13
76
2(A
C)1
0F
:C
CT
GC
AT
AA
TG
AG
CA
GC
AA
A
R:
TT
CT
TG
TA
AT
GC
CC
GT
GT
CA
41
64
–1
78
1.0
00
00
.65
25
0.0
02
60
.56
9
RC
19
JN4
13
76
3(C
A)7
(AC
)7F
:C
TC
TG
CT
TT
CA
GC
CT
CT
GC
T
R:
GC
AC
GT
AT
GT
AT
CT
GC
CT
TA
CA
31
87
–2
19
0.3
33
30
.29
43
0.7
03
10
.26
4
RC
20
JN4
13
76
4(A
C)2
6F
:T
GC
AT
GT
AA
GC
AT
TA
CG
TG
TT
T
R:
CG
AC
AT
CG
CT
TT
TT
GA
TC
CT
31
93
–2
08
0.8
56
40
.63
03
0.0
56
30
.54
4
RC
27
JN4
13
76
5(T
G)6
(GT
)5(G
T)5
(GT
)6F
:T
CC
CA
CT
CT
TC
TT
TG
TA
TT
TT
TG
A
R:
CA
GC
AG
CA
GG
AC
AA
AC
AC
TC
21
79
–1
85
0.7
91
70
.48
85
0.0
19
30
.36
4
RC
50
JN4
13
76
6(A
C)3
6F
:G
TC
TG
TG
CG
GT
CA
GT
GA
GA
A
R:
AG
CA
TT
CA
GA
GC
AG
GT
CA
GC
62
22
–2
46
1.0
00
00
.76
95
0.0
01
00
.71
8
RC
66
JN4
13
76
7(T
TA
GG
T)5
F:
TT
CC
TC
TA
AT
GT
TT
CC
TC
TA
AC
GT
G
R:
CC
CT
GA
CC
TG
AC
CC
TA
AC
AA
42
47
–2
53
0.4
16
70
.56
65
0.0
93
80
.47
2
RC
67
JN4
13
76
8(T
G)8
(TG
)5F
:G
CC
GT
TG
CA
TG
GC
TG
TA
T
R:
GA
CA
CA
CA
TC
GG
AT
TC
AT
GC
31
91
–2
07
0.5
00
00
.39
27
0.2
90
60
.32
5
RC
68
JN4
13
76
9(T
G)1
3F
:T
GA
AA
CC
AT
CT
GT
TG
CC
TG
A
R:
GA
GC
GT
TA
TC
TG
CC
AG
TG
TG
31
92
–2
10
0.7
08
30
.56
29
0.0
37
10
.46
6
RC
70
JN4
13
77
0(G
T)1
4F
:T
TG
AA
TG
AG
GA
CC
AG
CA
GT
G
R:
CG
TC
CT
CT
GT
CC
TT
TG
CT
TT
41
38
–1
52
0.9
16
70
.62
06
0.0
27
90
.52
8
RC
83
JN4
13
77
1(T
G)2
7F
:G
GT
CT
TG
GT
CT
TT
CT
CG
TC
AA
R:
GG
TT
CT
GG
GA
AA
TT
CT
TT
GG
A
41
98
–2
12
1.0
00
00
.59
84
0.0
07
90
.50
2
RC
86
JN4
13
77
2(A
C)6
(CA
)13
(AG
)6F
:C
CA
CT
AA
CC
CT
TC
CA
CT
TT
CT
CT
R:
CA
GC
AT
TG
AG
CT
GG
TA
TG
TA
CT
G
32
45
–2
51
0.8
75
00
.58
24
0.0
32
50
.49
6
RC
92
JN4
13
77
3(A
C)9
F:
GC
GA
CT
CA
TG
TG
AC
CA
GA
AA
R:
GA
TG
CC
CT
AT
TG
GA
CT
CG
TG
31
92
–2
10
0.9
58
30
.56
29
0.0
02
30
.45
4
RC
94
JN4
13
77
4(G
T)1
5F
:C
AT
GC
CT
TA
CA
CA
CA
CT
TC
CA
R:
CC
CA
CA
TT
GA
AG
GT
AG
TA
GA
AT
CA
61
69
–1
87
1.0
00
00
.64
27
0.0
00
10
.55
9
RC
10
2JN
41
37
75
(TG
)23
F:
TT
TA
TG
AA
GC
TG
TG
GT
GG
TG
R:
CC
AC
AT
TC
TT
CT
CA
GC
CA
AT
C
41
61
–1
73
1.0
00
00
.75
00
0.2
43
10
.68
5
RC
10
6JN
41
37
76
(GT
)8F
:G
AA
CA
GG
GA
GA
GA
GC
AG
GA
G
R:
TG
AC
CA
GC
CA
GG
AC
AT
AT
CA
31
57
–1
69
0.5
83
30
.67
02
0.5
23
70
.58
3
308 Conservation Genet Resour (2012) 4:307–310
123
screened using the SSRHunter software (Li and Wan
2005). One hundred and twenty-six positive clones were
sequenced and eighty sequences contained repeat motifs.
Forty-nine pairs of primers were designed using the
online software PRIMER version 3 (Rozen and Skaletsky
2000).
The polymorphism was evaluated using thirty samples
of R. cylindricus captured from the Zhuyangxi section in
the upper Yangtze River. PCR amplification was per-
formed in 10 ll reaction mixture which included 19
PCR buffer, 0.2 mM each dNTP, 2.0 mM MgCl2,
0.4 mM each primer, 0.5U Taq polymerase and about
20 ng template DNA. PCRs were performed on a PTC-
100TM thermocycler with the following conditions: 94�C
for 2 min followed by 35 cycles at 94�C for 30 s,
annealing at 58�C for 30 s, and at 72�C for 40 s, a final
extension at 72�C for 10 min. Amplified products were
analyzed on 10% non-denaturing polyacrylamide gel and
visualized by silver staining. Allele sizes were estimated
using a 10 bp ladder molecular size standard. The
observed (HO) and expected (HE) value for heterozy-
gosity, deviations from Hardy–Weinberg equilibrium
(HWE) and linkage disequilibrium were calculated by
POPGENE version 1.31 (Yeh et al. 1999). Polymorphism
information content (PIC) was estimated by PIC-CALC
version 0.6.
Of the 49 primer pairs trialed, 36 produced discrimi-
nable PCR products and the remaining 13 resulted in poor
or no amplification in R. cylindricus. Within the 36 loci
that have been successfully amplified, 19 loci exhibited
polymorphic and the rest 17 were miscellaneous or
monomorphic. The characteristics of the 19 polymorphic
microsatellite loci were shown in Table 1. The number of
alleles, observed and expected heterozygosity ranged from
2 to 6, from 0.3333 to 1.000, and from 0.2943 to 0.7695,
respectively. These polymorphic loci exhibited relatively
high observed heterozygosity. The PIC ranged from 0.264
to 0.718. Ten loci were highly informative (PIC [ 0.5),
nine were moderately informative (0.25 \ PIC \ 0.5)
(Botstein et al. 1980). Six loci (RC11, RC18, RC50,
RC92, RC94 and RC119) significantly deviated from
HWE after Bonferroni correction (adjusted P \ 0.004),
which might be caused by null alleles or Wahlund effect.
Two groups of couple loci (RC11 and RC110, RC20 and
RC68) exhibited significant linkage disequilibrium. These
polymorphic microsatellite markers will be useful for
studying population structure, gene flow and conservation
genetics of R. cylindricus.
Acknowledgments We thank Cunyao Li and Geng Chen for the
technical assistance. This work was supported by the National Sci-
ence Foundation (51109091), Special Fund for Agro-scientific
Research in the Public Interest of China (200903048) and the Fund of
Wuhan Bureau of Education (2010060).Ta
ble
1co
nti
nu
ed
Lo
cus
Acc
essi
on
no
.R
epea
tm
oti
fP
rim
er(50 –
30 )
AS
ize
ran
ge
(bp
)H
OH
EP
PIC
RC
11
0JN
41
37
77
(CA
)8F
:G
TG
TC
AA
CA
TG
GC
AC
AA
CA
A
R:
CG
AG
GG
CG
AA
TA
AA
TA
AC
GA
21
53
–1
61
0.8
58
30
.50
98
0.0
10
70
.37
5
RC
11
9JN
41
37
78
(TG
)13
F:
TG
AC
TC
TG
CA
GT
GA
GG
CT
TG
R:
TA
GC
CA
GC
TA
CG
CA
CT
AC
GA
41
25
–1
37
1.0
00
00
.65
25
0.0
00
10
.56
9
An
um
ber
of
alle
les,
HO
ob
serv
edh
eter
ozy
go
sity
,H
Eex
pec
ted
het
ero
zyg
osi
ty,
Pex
act
Pv
alu
efo
rth
eH
WE
test
,P
ICp
oly
mo
rph
ism
info
rmat
ion
con
ten
t
Conservation Genet Resour (2012) 4:307–310 309
123
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