4
TECHNICAL NOTE A first set of polymorphic microsatellite loci isolated from 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 Ó 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: 5 0 -TAC TCA GGA CTC AT-3 0 and MseIB: 5 0 -GAC GAT GAG TCC TGA G-3 0 ). 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: duan@yfi.ac.cn 123 Conservation Genet Resour (2012) 4:307–310 DOI 10.1007/s12686-011-9531-3

A first set of polymorphic microsatellite loci isolated from Rhinogobio cylindricus

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Page 1: A first set of polymorphic microsatellite loci isolated from Rhinogobio cylindricus

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

Page 2: A first set of polymorphic microsatellite loci isolated from Rhinogobio cylindricus

Ta

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TG

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21

59

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12

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61

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CG

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TT

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31

93

–2

08

0.8

56

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0.0

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.54

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JN4

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G)6

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T)5

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22

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1.0

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0.4

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2

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TG

31

92

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10

0.7

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30

.56

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.46

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RC

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TG

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41

38

–1

52

0.9

16

70

.62

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0.0

27

90

.52

8

RC

83

JN4

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1(T

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CT

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CT

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R:

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TT

CT

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41

98

–2

12

1.0

00

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.59

84

0.0

07

90

.50

2

RC

86

JN4

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77

2(A

C)6

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TG

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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

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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

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TT

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GG

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TC

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GC

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AT

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41

61

–1

73

1.0

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10

.68

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(GT

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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

Page 3: A first set of polymorphic microsatellite loci isolated from Rhinogobio cylindricus

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

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ICp

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ism

info

rmat

ion

con

ten

t

Conservation Genet Resour (2012) 4:307–310 309

123

Page 4: A first set of polymorphic microsatellite loci isolated from Rhinogobio cylindricus

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