MICROSATELLITE LETTERS

Isolation and characterisation of microsatellite loci in the tiretrack eel, Mastacembelus favus and cross-species amplification

Amirul Firdaus Jamaluddin Jamsari •

So Nam • Bui Minh Tam • Mohd Nor Siti-Azizah

Received: 25 November 2013 / Accepted: 25 December 2013 / Published online: 31 December 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Nine microsatellite loci were isolated and

characterized in the tire track eel, Mastacembelus favus by

using library enriched for microsatellite region. Among 47

specimens collected from Mekong River, the number of

alleles ranged from 2 to 17 while levels of observed and

expected heterozygosities ranged from 0.021 to 0.873 and

from 0.021 to 0.893, respectively. The primers were suc-

cessfully tested in six closely related species, hence valu-

able for investigating population genetics and other related

aspects of M. favus and its congener species.

Keywords Mastacembelus favus � Enriched library �Microsatellites � Cross-amplification � Conservation

The tire track eel, Mastacembelus favus (Mastacembelidae)

is a widely distributed tropical freshwater fish across

Thailand, Lao PDR, Cambodia, Vietnam and Peninsular

Malaysia. It is considered as an excellent food fish and

sometimes seen in the aquarium trade (Jamsari pers. obs.;

Froese and Pauly 2013). In contrast to its significant value,

information on its basic ecology and biology i.e. migratory

pattern, spatial and temporal patterns of population

dynamics and evolutionary trends is still scarce or

unknown. Therefore, to aid the monitoring of the species,

development of novel microsatellites from M. favus and

cross-amplification of six congeners were conducted.

Genomic DNA was extracted from a single individual of

M. favus using AquaGenomic DNA isolation Kit (Bio-

Syntech) and microsatellite-enriched library protocol as

described in Jamsari et al. (2011). Fifty-one positive col-

onies were selected for sequencing. However, only 17

sequences exhibited good signal data with at least eight

repeat microsatellite motifs and primers were designed

using Primer3 (Untergasser et al. 2007). Preliminary

assessment on several individuals of M. favus with opti-

misation of annealing temperatures (40–65 �C) indicated

only nine loci with successful amplifications. These were

subsequently labelled with fluorescent dye for multiplex

PCR (Table 1).

Forty-seven individuals of M. favus collected from

Mekong River were then assayed for the nine microsatellite

characterisation. The loci were arranged in two 25 ll

multiplex PCR amplifications and unless otherwise stated,

PCR protocol was as described in Mytaq Red DNA poly-

merase (Bioline) standard protocol. Allelic data was ana-

lyzed using PeakScanner v1.0 software (Applied

Biosystems) with GS500 (Applied Biosystems) as the

internal size standard.

Results generated from Arlequin version 3.1 (Excoffier

et al. 2005) showed that the number of alleles per locus for

polymorphic loci ranged from 2 to 17 with a mean of 7.4.

The observed and expected heterozygosities ranged from

0.021 to 0.873 and from 0.021 to 0.893, respectively. All

A. F. J. Jamsari (&) � M. N. Siti-Azizah

School of Biological Sciences, Universiti Sains Malaysia,

11800 Penang, Malaysia

e-mail: jamsari_85@yahoo.com

S. Nam

Inland Fisheries Research and Development Institute (IFReDI),

Fisheries Administration, 186, Norodom Blvd., Phnom Penh,

Cambodia

B. M. Tam

College of Aquaculture and Fisheries, Can Tho University,

Can Tho, Vietnam

M. N. Siti-Azizah

Centre for Marine and Coastal Studies (CEMACS-Muka Head),

Universiti Sains Malaysia, 11800 Penang, Malaysia

123

Conservation Genet Resour (2014) 6:477–479

DOI 10.1007/s12686-013-0132-1

Ta

ble

1C

har

acte

riza

tio

no

fn

ine

mic

rosa

tell

ites

for

47

ind

ivid

ual

so

fti

retr

ack

eel,

Ma

sta

cem

bel

us

favu

sco

llec

ted

fro

mM

eko

ng

Riv

er

Lo

cus

Pri

mer

seq

uen

ce(50 –

30 )

Mu

ltip

lex

set

Rep

eat

mo

tif

Gen

Ban

kac

cess

ion

no

.A

llel

esi

ze(b

p)

Ta

(�C

)M

eko

ng

Riv

er

(n:4

7)

NA

HO

HE

P

MF

01

-E0

7F

:GG

GT

CC

AC

GA

GT

GT

TC

AA

AT

1(T

G) 1

1K

F8

00

88

01

92

–1

98

40

–5

55

0.5

96

0.6

11

0.1

61

8

R:H

EX

-CA

AG

GG

CT

CT

CC

AG

TG

TC

AT

MF

01

-D0

7F

:HE

X-C

AC

CA

GC

AA

CC

TG

CA

TT

TT

T1

(CA

) 22

KF

80

08

81

11

9–

15

14

5–

55

11

0.7

45

0.8

48

0.0

93

3

R:A

TG

TA

TG

CA

GC

CT

GG

AC

CT

T

MF

03

-P1

0F

:FA

M-T

GA

CC

AG

AG

GT

CA

AG

CA

GA

A1

(CA

) 11

KF

80

08

82

24

9–

25

95

0–

60

20

.02

10

.02

11

.00

00

R:C

CG

TC

CT

CC

AC

AG

GT

AG

AC

A

MF

02

-P0

4F

:GT

GG

AC

TA

AC

AG

AG

GC

AT

TC

G1

(CA

) 26

KF

80

08

83

27

3–

32

74

0–

50

17

0.8

73

0.8

93

0.2

82

3

R:H

EX

-CG

TG

GA

CT

AG

GT

GA

CG

TG

AA

MF

01

-H0

5F

:HE

X-C

GG

TG

AA

GA

CA

CT

CG

CA

TA

A2

(CA

) 17

KF

80

08

84

10

1–

13

04

0–

55

10

0.7

45

0.7

75

0.6

29

3

R:A

CT

TC

CT

CC

AC

CT

CC

GT

TT

T

MF

01

-F0

6F

:FA

M-C

GG

CT

GA

AT

GT

TA

CC

AC

TT

G2

(CA

) 10

KF

80

08

85

14

1–

14

54

0–

55

30

.51

10

.45

40

.78

66

R:C

AT

TC

GC

AG

TA

AA

AC

GC

AG

A

MF

01

-A0

6F

:GG

CC

TC

TT

CT

GA

CA

CT

CT

GC

2(C

A) 1

5K

F8

00

88

61

89

–1

99

40

–5

06

0.6

38

0.6

26

0.7

49

4

R:H

EX

-CC

AC

AG

GA

CA

TG

GA

CC

TT

CT

MF

01

-B0

7F

:HE

X-C

GA

TG

AA

GA

GG

AG

GC

TT

GA

C2

(TG

) 8A

GK

F8

00

88

72

77

–3

14

40

–5

57

0.7

02

0.6

41

0.9

75

9

R:C

CT

CC

AC

TC

TG

GT

CG

AT

GT

T(T

G) 1

0

MF

02

-P0

8F

:FA

M-C

CA

CA

GA

AA

CT

TC

CC

CT

GA

A2

(CA

) 9A

AK

F8

00

88

82

02

–2

16

40

–5

56

0.6

81

0.6

93

0.6

43

2

R:T

GT

GG

GT

TG

AT

GT

AG

GC

TG

A(C

A) 4

All

ele

size

ran

ge

inb

ase

pai

rs(b

p);

ann

eali

ng

tem

per

atu

re(T

a);

nu

mb

ero

fal

lele

s(N

A);

ob

serv

edh

eter

ozy

go

sity

(HO

);ex

pec

ted

het

ero

zyg

osi

ty(H

E);

Pv

alu

e(P

)

478 Conservation Genet Resour (2014) 6:477–479

123

loci conformed to Hardy–Weinberg equilibrium with no

evidence of linkage disequilibrium. Analysis using Mi-

croChecker v2.2.3 (Van Oosterhout et al. 2004) indicated

no evidence for null alleles, large allele dropout, and

scoring error due to stuttering. The potential of cross-

amplification was also tested for six Asian Mastacembelus

species. Relatively high level of cross-species amplification

was observed ranging from 4 to 8 loci (Table 2).

Overall, high variable microsatellite markers developed

in this study would be useful in assessing the patterns of

population genetic structure, genetic diversity, history

biogeography, breeding strategy, fishery management and

formulating conservation priorities of this and closely

related species.

Acknowledgments We would like to acknowledge Universiti Sains

Malaysia for research funding (Research University Grant—1001/

PBIOLOGI/815061) and for Vice Chancellor’s Award to the first

author for his Ph.D. program. We would like to thank Phanara Thach,

Nguyen Hong Quyet Thang and Dang Quang Hieu for their assistance

in collecting the samples.

References

Excoffier L, Laval G, Schneider S (2005) ARLEQUIN (v. 3.11): an

integrated software package for population genetics data

analysis. Evol Bioinform Online 1:47–50

Froese R, Pauly D (eds) (2013) FishBase. World Wide Web electronic

publication. http://www.fishbase.org, version (10/2013). Acces-

sed 4 October 2013

Jamsari AFJ, Min Pau T, Siti Azizah MN (2011) Isolation and

multiplex genotyping of polymorphic microsatellite DNA

markers in the snakehead murrel, Channa striata. Genet Mol

Biol 34:345–347

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen

JA (2007) Primer3Plus, an enhanced web interface to Primer3.

Nucleic Acids Res 35(Web Server issue):W71–W74

Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004)

Micro-checker: software for identifying and correcting genotyp-

ing errors in microsatellite data. Mol Ecol Notes 4:535–538

Ta

ble

2C

ross

amp

lifi

cati

on

resu

lts

for

six

spec

ies

of

mas

tace

mb

elid

s

Sp

ecie

sL

ocu

s

MF

01

-E0

7M

F0

1-D

07

MF

03

-P1

0M

F2

-P0

4M

F0

1-H

05

MF

01

-F0

6M

F0

1-A

06

MF

01

-B0

7M

F2

-P0

8

Ma

sta

cem

bel

us

arm

atu

s?

-?

-?

??

??

Ma

sta

cem

bel

us

alb

og

utt

atu

s-

DB

?W

A?

?W

AW

A?

Ma

sta

cem

bel

us

no

top

hth

alm

us

??

?D

B?

??

??

Ma

sta

cem

bel

us

un

ico

lor

-?

??

?-

??

?

Ma

sta

cem

bel

us

eryt

hro

taen

ia-

??

??

??

??

Ma

sta

cem

bel

us

tin

win

i?

WA

??

??

?D

B?

Su

cces

sfu

lam

pli

fica

tio

n(?

);w

eak

amp

lifi

cati

on

(WA

);d

ou

ble

ban

dam

pli

fica

tio

n(D

B);

no

amp

lifi

cati

on

(-)

Conservation Genet Resour (2014) 6:477–479 479

123