fenneropenaeus indicus 81 microsatellite markers

8/18/2019 Fenneropenaeus Indicus 81 Microsatellite Markers

1/8

c Indian Academy of Sciences

ONLINE RESOURCES

Development and characterization of eighty-one microsatellite markers

in Indian white shrimp, Fenneropenaeus indicus, through

cross-amplification

K. A. SAJEELA1∗, A. GOPALAKRISHNAN2, V. S. BASHEER 1, K. K. BINEESH1 and J. K. JENA3

1 National Bureau of Fish Genetic Resources Kochi Unit, and 2 Central Marine Fisheries Research Institute,

Cochin 682 018, India3 National Bureau of Fish Genetic Resources, Canal Ring Road, Lucknow 226 002, India

[Sajeela K. A., Gopalakrishnan A., Basheer V. S., Bineesh K. K. and Jena J. K. 2015 Development and characterization of eighty-onemicrosatellite markers in Indian white shrimp, Fenneropenaeus indicus, through cross-amplification. J. Genet. 94 , e43–e50. Online only:http://www.ias.ac.in/jgenet/OnlineResources/e43.pdf ]

Introduction

Indian white shrimp, Fenneropenaeus indicus, is an impor-

tant crustacean species in the commercial fish landings of

southwest and southeast coasts of India. It also forms a major

fishery in African coast (Mozambique, Tanzania and Kenya),

Sri Lanka, Red Sea and Persian Gulf. To reveal the genetic

stock structure and gene mapping studies of F. indicus, we

developed 81 polymorphic microsatellites through cross-

amplification after screening 396 primer pairs from other

penaeids. This genetic information will be of immense use inmanagement of stocks and selective breeding programmes of

F. indicus.

The wild populations of F. indicus from different parts of

the world may be distinct with respect to phenotypic traits

such as growth, fecundity, feed conversion efficiency, salin-

ity tolerance and disease resistance. Determination of genetic

variation in natural populations of commercially important

fishes would help in identifying their genetic strains, if any.

In F. indicus, information of the same can be used for its

genetic upgradation, fisheries management and conserva-

tion programmes. Molecular genetic markers like microsatel-

lites are useful in studying the genetic variability of natural

populations (at intraspecific level). Greater the number of

microsatellite markers available easier it is to construct the

linkage map of a species which would help the breeders to tag

the desired genes and consequently breed cultured shrimps.

This, points to the need to develop more microsatellite

markers for different species.

In the present study, we developed 81 microsatellite mark-

ers through cross-species amplification from related species

∗For correspondence. E-mail: [email protected].

which can be helpful in unraveling the genetic structure

among the wild stocks of F. indicus.

Materials and methods

For cross-species amplification, altogether a total of 396

primer pairs were identified from different penaeids from

published papers (Xu et al. 1999; Wang et al. 2005; Dong

et al. 2006; Zhi-Ying et al. 2006; Freitas et al. 2007; Gao

et al. 2008; etc.) and from NCBI GenBank accessions (http://www.ncbi.nlm.nih.gov ). The cross-species amplification tri-

als were done for 20 to 30 specimens of F. indicus in different

size groups (100–200 mm in total length), collected from the

trawl landings at fisheries harbour, Cochin during September–

December 2010. After recording the total length, carapace

length, total weight and sex of the specimens, the total

DNA was extracted from the gills of the samples, follow-

ing salting out procedure of Miller et al. (1988). Amplifi-

cations were performed in VeritiTM 96-Well Thermal Cycler

(Applied Biosystems, Carlsbad, USA) using standardized

protocols. PCR reactions were carried out in 25 µL reac-

tion mixture containing 1× reaction buffer (10 mM Tris, 50mM KCl, 0.01% gelatin and pH 9.0) with 1.5 mM MgCl 2(Genei, Bengaluru, India), 5 pmol of each primer, 200 mM

dNTPs, 2 U Taq DNA polymerase (Fermentas, Burling-

ton, Canada) and 25–50 ng of template DNA. The reac-

tion mixture was preheated at 94◦C for 5 min followed by

25 cycles (94◦C for 30 s, annealing temperature depending

upon the T m value of primer (usually 50–60◦C, see table 1)

and 72◦C for 2 min). The reaction conditions were standardized

for different primers for fine results. The amplified products

were electrophoretically analysed through 10% nondenaturing

Keywords. cross priming; microsatellites; Indian white shrimp.

Journal of Genetics Vol. 94, Online Resources e43

http://www.ias.ac.in/jgenet/OnlineResources/e43.pdfhttp://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ias.ac.in/jgenet/OnlineResources/e43.pdf


2/8


3/8

Characteristics of microsatellite loci in F. indicus

T a b l e 1

( c o n t d )

F .

i n d i c u s

R e s o u r c e

S i z e

T a

S i z e

T a

A c c e s s i o n

s p e c i e s

L o c u s

P r i m e r s e q u e n c e ( 5 – 3 )

R e p e a t m o t i f

( b p ) ( ◦ C )


( b p )

( ◦ C ) N a H

o

H e

n u m b e r

2 2

L . v a n n a m e i L v a n 1

F : C C C T

T T A C C A C C T C C T T C A A T C

( C T ) 3

1 6 6

5 0

( C T ) 8

1 8 0 – 1 9 0

5 0

2

0 . 2 0 4

0 . 2 1 3

J F 7 1 5 2 1 8

R : A A G A G G A G G G G A A G G G T C A G

2 3

L v a n 2

F : C C A T

G G C T T T C C T C T T C T T T C

( T C C

) 5 . . . ( C C T ) 3 . . .

3 2 7

5 0

( T C C ) 7 . . . ( C C T ) 6

3 0 9 – 4 0 4

5 0

4

0 . 3 9 7

0 . 5 2 1

J F 7 1 5 2 5 9

R : A G G T A G G G A A G T C G T G A G G G

( C C T

) 3 . . . ( T C ) 4 . . . ( T C ) 4

2 4

L v a n 3

F : T G T C

G T T A G T G C A G C T C A T T C

( T T C ) 3 T T ( T T C ) 3 . . .

1 7 6

5 0

( T T C ) 1 0

2 0 1

5 0

3

0 . 2 6 4

0 . 3 1 2

J F 7 1 5 2 6 0

R : G G G G A G G A A T A A G A G G A A A G G

( T C C

) 3 C ( T C C ) 5

2 5

L v a n 4

F : G G C A C A C T G T T T A G T C C T C G

( G T T

) 3 . . . ( G A ) 3 . . . ( T C ) 3 . . . 2 4 2

5 0

( T C ) 8

2 4 2

5 0

3

0 . 3 0 2

0 . 3 6 5

J F 7 1 5 2 6 1

R : C G A A C A G A A T G G C A G A G G A G

( G T ) 3 . . . ( T C ) 3 . . . ( T C ) 3

2 6

L v a n 5

F : A G A C A C A T A C A G A C G C A C G C

( A C ) 3

. . . ( A C ) 3 . . .

3 2 9

5 0

( C A ) 1 3 . . . ( C A ) 6

3 0 9 – 4 0 4

5 0

3

0 . 3 4 2

0 . 4 0 3

J F 7 1 5 2 6 3

R : G A G T T G C T C C C A A A C G C T A C

( C A ) 1 9 . . . ( C A ) 7 . . .

2 7

L v a n 1 3

F : G A G A G C A A A T A A G A A A G G G C

( G G A

) 3 ( G A ) 3 . . . ( C T ) 3 C C

2 1 9

5 0

( G G

A ) 4 . . . ( T C C C ) 3 . . . 1 8 0

5 0

1 1 0 . 4 3 2

0 . 6 8 3

J F 7 1 5 2 2 8

R : A G G A T G C A A A T G A T A A C G A G

( C T ) 3 C C ( C C C T ) 6 ( C T ) 1 0

( T C ) 9

2 8

L v a n 6

F : C A C A

T C A T G T C A C T G C T A C G A C

( A T ) 3

2 3 4

5 0

( A T ) 8

3 0 9 – 4 0 4

5 0

2

0 . 1 9 7

0 . 3 5 1

J F 7 1 5 2 5 3

R : G C T G C A C A A T C A A C T T G C T T A C

2 9

L v a n 7

F : G A A T

G G G A G G A G A A G G A T A G

( A A C

) 3 ( A G ) 3 . . . ( T ) 2 6

1 0 5

5 3

( A G

) 3 . . ( T ) 1 8

1 2 3

5 3

2

0 . 1 6 8

0 . 2 2 4

J F 7 1 5 2 6 2

R : T T C C

A C G T G G T T T C C C G A T G

3 0

L v a n 8

F : G A G A A G A G G C T G C T T T G T C G

( T A ) 3

C A A ( A T ) 3

2 7 8

5 0

( T A ) 7

2 4 2 – 3 0 9

5 0

2

2 1

2

0 . 3 2 1

J F 7 1 5 2 3 4

R : T G A C T T T G A A C T G G T G T G C G

3 1

L v a n 9

F : G A C G A A C A G C C A G T C A A C C

( T C ) 4 . . . ( T C ) 4 . . . ( T C ) 1 4 . . .

2 8 8

5 0

( T C ) 1 4

2 4 2 – 3 0 9

5 0

4

0 . 3 6 5

0 . 4 1 2

J F 2 9 7 6 5 6

R : G G G G A T A G G G T A G C G G A A G

( T C ) 3

3 2

L v a n 1 0

F : A T T C

T T T G T G T T T C T T C G C C

( C A ) 3

. . . ( G T ) 5 . . .

1 1 3

5 1

( G T ) 9

1 4 7 – 1 6 0

5 0

2

0 . 1 8 6

0 . 2 6 1

J F 7 1 5 2 2 0

R : C G T C C C T G A A A C T T T A T C T C C

( G T ) 3 . . .

3 3

L v a n 1 1

F : A G A G T C C T T G G T G A G T A G C

. . . ( T C ) 1 1 T T T T C T A T A

3 5 3

5 3

( T C ) 1 5

3 0 9 – 4 0 4

5 0

3

0 . 2 9 5

0 . 3 6 1

J F 7 1 5 2 2 1

R : G A G C G A T A G A G T G C A A T A A A G

( T C ) 1 1

3 4

L v a n 1 2

F : A C A C A C C C A T C C A A C T A C C C

( C A ) 3 5 A A C ( A C G C ) 4 . . .

3 2 1

5 5

( C A ) 1 7 . . ( A C C C ) 3 . .

< 3 0 9

5 0

4

0 . 4 3 5

0 . 4 9 8

J F 7 1 5 2 1 9

R : G G C C T A T G G T T T G T C T G A G G

( C A ) 3

A ( A C C C ) 3 ( A C ) 3

( C A C G ) 4

3 5

L v a n 0 5 1 2

F : T G C C

A G T G C C A T T T G A

( T A T ) 4 T T T ( T A T ) 2

2 5 8

5 0

( T A T

) 6

3 0 9

5 0

7

0 . 4 4 7

0 . 8 9 9

J N 1 8 5 4 2 8

R : C C T C

C T C C T C C C A A C T

3 6

P v a n 1 4

F : C T C C

A G G A C C G A T A A T G A G G

( T C ) 3 . . . ( T C G ) 3 . . .

1 1 8

5 5

( T C G ) 3 . . . ( T C ) 2 3

4 0 4

5 5

4

0 . 5 6 2

0 . 5 9 7

J N 1 8 5 4 2 9

R : C G A C A G T C A A A A C A A A C A T C C

( T C ) 2 5

3 7

P v a n 1 5

F : C T A C

T T A T C G G T C T T T C T A C T T A C C

( T G ) 4 ( C G ) 3 . . . ( A C ) 7

2 0 6

5 5

( C A ) 2 4

2 4 2 – 2 3 8

5 5

2

0 . 1 4 2

0 . 2 6 8

J N 1 8 5 4 3 0

R : C T T A

G T G T T T T G T T C A C C C C

( A C G

C ) 3 G C ( A C ) 2 8 . . .

3 8

L v a n 0 1

F : T G T C

T G A A G A G G G A C T C G T G

( G T ) 6 . . . ( A C ) 3 ( A T ) 5 . . .

1 8 0

5 0

( G T ) 6 . . . . ( A T ) 2 4

1 6 0 – 1 8 0

5 5

6

0 . 5 8 4

0 . 6 3 2

J F 7 1 5 2 3 8

R : T T G T

G C A T T G T G G G T T T T T C

( A T ) 2 7

3 9

L v a n 0 5 1

F : G A G T T C C A A T G T A A G T A G

( A ) 7 T

( A ) 3 G ( A ) T ( A ) 4 T ( A ) 4

1 2 4

5 0

( A ) 7 T ( A ) 3 G ( A ) T

1 2 3 – 1 4 7

5 5

2

0 . 1 3 9

0 . 3 1 1

J F 7 1 5 2 3 9

R : A A A A T G T A G G T C G G T C

( A ) 4 T ( A ) 4

4 0

L v a n 0 5 2

F : A G C C A G G A A G A G G A G G

( G A G

C ) 4

1 1 2

5 0

( T ) 6 . . . . ( T ) 6

1 1 0 – 1 2 3

5 5

2

0 . 2 1 1

0 . 3 2 5

J F 7 1 5 2 4 0

R : C A T C

G C C A G A A A G A C A G

4 1

L v a n 0 5 3

F : T T A C

G G G T G A A G T G T T

( A C ) 7

2 8 9

5 5

( A C ) 8

3 0 9 – 4 0 4

5 5

2

0 . 1 7 2

0 . 2 4 1

J F 7 1 5 2 4 1

R : T T T A

T G C T T C C C T A C C



4/8


5/8


T a b l e 1

( c o n t d )

F .

i n d i c u s

R e s o u r c e

S i z e

T a

S i z e

T a

A c c e s s i o n

s p e c i e s

L o c u s

P r i m e r s e q u e n c e ( 5 – 3 )


( b p )

( ◦ C )

R e

p e a t m o t i f

( b p )

( ◦ C )

N a

H o

H e

n u m b e r

6 3

P . m o n o d o n

P M C 2 8 1

F : G G C A G G A A T G T C A A C C A A A T

T

1 5

1 7 1 – 1 7 5

5 5

( T ) 1 1

1 6 0 – 1 8 0

5 5

5

0 . 6 2 1

0 . 7 5 1

J N 1 8 5 4 3 1

R : C C G

G G T A T A A A C T A C A C A T C A A A A C

6 4

P M C 3 1 1

F : C C A T C A A A G T A A A T C A G A A C C A G A G

( A A T ) 4

1 1 5 – 1 1 8

5 5

( T A A A ) 6

2 0 1 – 2 1 7

5 5

2

0 . 1 7 5

0 . 2 8 6

J N 1 8 5 4 3 2

R : T G A

G T C T T G C A G C T C G A A A A T A

6 5

P M 1 3 8

F : A C G G A G T G G G T A G A G A C A T A

( G T ) 4 7

2 6 8 – 3 3 8

5 6

( G T ) 4 0

1 2 3 – 1 4 7

5 6

2 2

0 . 7 4 3

0 . 8 6 7

J N 1 8 5 4 1 8

R : A C A

A G C G A A G T G A A G A G G

6 6

P M 2 0 5

F : A G G

A A T G A T G G G A G G G A A A G

( A G ) 2 3

1 5 3 – 2 0 9

5 6

( C A

) 3 3

1 4 7 – 1 6 0

5 6

1 7

0 . 6 5 8

0 . 8 4 5

J N 1 8 5 4 1 9

R : A A G

C T C A G G C A A G C G T G T A T

6 7

P M 5 8 0

F : A A C T G C C T A C A G T G T G T G C G

( A G ) 2 8

2 5 0 – 3 4 0

5 6

( G A

) 2 9

1 4 7 – 1 6 0

5 6

2 3

0 . 8 8 9

0 . 9 3 4

J N 1 8 5 4 2 0

R : G A A

T G G A G C C T G T T G G T T T G

6 8

P M 2 3 4 5

F : G A T A

T T T C A A G G A A T G C T C G

( T C ) 4 2

1 4 3 – 2 2 9

5 6

( T C ) 3 2

1 6 0 – 1 8 0

5 6

2 0

0 . 6 7 5

0 . 8 3 9

J N 1 8 5 4 2 1

R : T A A T T C G T G C C T T A C C T C A T

6 9

P M 3 5 3 8

F : G A A

C G T C G G G G G A T T T A C T T

( A C ) 1 2

3 7 1 – 4 4 1

5 6

( C A

) 1 8

2 4 2 – 3 0 9

5 6

1 7

0 . 7 3 2

0 . 9 2 1

J N 1 8 5 4 2 2

R : A C T A T C A C A C C G A G G C T T G G

7 0

P M 3 8 5 4

F : T C T T

G G T C G G A A T G G G T A A G

( G T ) 1 6 . . . ( G T ) 3 3

1 8 4 – 3 1 6

5 6

( G T ) 3 7

1 2 3 – 1 8 0

5 6

1 8

0 . 6 6 8

0 . 8 4 5

J N 1 8 5 4 2 3

R : T T C T G A G A A G G C A C A C A T G C

7 1

P M 4 0 1 8

F : G T T C C A A G C G A C A G A C G A G T

( A C ) 2 7

1 7 7 – 2 5 5

5 4

( A C

) 2 4

2 1 7 – 2 4 2

5 4

2 0

0 . 7 8 7

0 . 9 2 2

J N 1 8 5 4 2 4

R : C G A

A T G C A C T G C C T G T A T G T

7 2

P M 4 0 8 9

F : C T T T

T T G A A A T C G C C C T G T T

( C A ) 4 4

2 4 3 – 3 7 7

5 6

( C A

) 3 9

2 4 2 – 3 0 9

5 6

2 5

0 . 8 2 5

0 . 9 0 6

J N 1 8 5 4 2 5

R : C A T T C A T C C C G C T C T T C T G T

7 3

P M 4 7 9 8

F : G C T T G C G T G T G T G C A T A C T T

( T G ) 3 2 . . . ( T G ) 1 6

2 7 5 – 4 3 1

5 2

( T G ) 2 7 . . . . ( T G ) 2 4

2 4 2 – 3 0 9

5 2

2 6

0 . 8 6 7

0 . 9 3 1

J N 1 8 5 4 2 6

R : G T T C C C C T C G T G T T T A C G A A

7 4

P M 4 8 5 8

F : G C C T T G T T A C G G T G G A G G T A

( A C ) 1 6

2 1 5 – 2 9 5

5 5

( C A

) 1 2

2 4 2 – 3 0 9

5 5

2 3

0 . 6 2 8

0 . 8 6 8

J N 1 8 5 4 2 7

R : C G G

C C T A T A A C T G T C T G C C T

7 5

P M 4 9 2 7

F : G G G

G A A T T A A T C T G C C C A T T

( C A ) 2 5

2 9 6 – 3 6 2

5 3

( A C

) 4 0

1 6 0 – 1 8 0

5 3

2 0

0 . 7 9 1

0 . 9 2 1

J N 7 8 7 9 6 0

R : A A T G G C A C A A G C A A A A G G A C

7 6

P M 5 2 1 3

F : T G G A C T G A G G T A T G C A G C A C

( A T ) 6 . . . ( C A ) 1 9

2 3 1 – 2 8 3

5 3

( A T ) 7 ( A C ) 1 8

2 4 2 – 3 0 9

5 3

1 5

0 . 6 8 7

0 . 7 8 2

J N 7 8 7 9 6 1

R : T C C T T G T T T G G A A C C C T T T G

7 7

P m o 2 5

F : G G T G C G T G T T T G T C G T A A A T A C T G G C ( T G ) 2 1

1 3 2 – 2 0 6

5 3

( T G ) 1 5

< 2 4 2

5 0

2 6

0 . 8 4 5

0 . 8 9 6

J F 7 1 5 2 2 6

R : C A T G C C C T T C C T T G A C G C C A A C C C T C

7 8

C U 4 6

F : T G T G T A A C A G C C T T C C C T G T G C

E

S T - S S R

2 9 5

5 5

( A T ) 6

3 0 9 – 4 0 4

5 0

4

0 . 2 5 8

0 . 3 4 5

J N 7 8 7 9 5 7

R : T T T A

G C C A A C T A C C T G G A C A A G C

7 9

C U 7 3

F : T C T C

A A G C A T A T C C A C G G G

E

S T - S S R

2 2 6

5 5

( T G ) 6

2 0 1 – 2 1 7

5 0

7

0 . 4 5 6

0 . 6 5 8

J N 7 8 7 9 5 8

R : A A C

A C G T C A T C A C A A G C T G C

8 0

C U 1 3 5

F : C C T T C T T G G T G C T G T G A C T G

E

S T - S S R

1 7 8

5 5

( C C C C G ) 5

1 6 0 – 1 8 0

5 0

6

0 . 5 2 4

0 . 6 2 8

J N 7 8 7 9 5 9

R : G C C

T T C G T T T A T C G C T T G T C

8 1

S A L 9 6

F : G A A

G G T G A T G G T G G G T T C C

E

S T - S S R

1 4 3

5 5

( G A

C T ) 4

1 6 0 – 1 8 0

5 0

2

0 . 1 4 6

0 . 4 2 1

J N 9 7 7 1 3 9

R : T C T A A G C G G G G A C T A A C A G C

T a , a n n e a l i n g t e m p e r a t u r e ; N a , n u m b e r

o f a l l e l e s .



6/8

K. A. Sajeela et al.

polyacrylamide gel (19 : 1 acrylamide : bisacrylamide) and

visualized through silver staining. The alleles were

designated according to PCR product size relative to a

known molecular weight ladder ( pBR322DNA/ MspI digest).

To confirm the occurrence of repeats, all the cross-amplified

polymorphic microsatellite loci were analysed by cloning in

TOPO vector (Invitrogen, Carlsbad, USA) and sequencing

in forward and reverse directions. The sequencing was done

with the automated DNA sequencing ABI Genetic Analyzer

3730 platform (Applied Biosystems, Carlsbad, CA).

The data were analysed using software Genetix 4.02

(Belkhir et al. 1997) to obtain allele frequencies, mean num-

ber of alleles per locus, expected ( H e) and observed ( H o) het-

erozygosity values. Tests for conformity to Hardy–Weinberg

expectations (HWE) were performed using Markov chain

method with parameters dememorization = 1000, batches =

100 and iteration = 100 (GenePop 4.1.1, Rousset 2008). The

data was also analysed for genotype linkage disequilibrium

between pairs of loci in a population based on null hypoth-

esis (genotypes at one locus is independent of genotypesat other loci), using the Genepop 4.1.1 programe (Rousset

2008). The genotypes of the loci deviating from HWE were

tested according to Van Oosterhout et al . (2004, 2006)

using MICRO-CHECKER for genotyping errors because of

nonamplified alleles (null alleles).

Results and discussion

Screening of 396 microsatellite primers generated 102

successful amplifications (25.76%) for the target species

in standard PCR conditions. After sequencing of 716

clones (102 loci in seven individuals each), 81 loci

(20.45%) were confirmed as microsatellites with repeat

sequences and 21 loci were nonrepeating EST mark-

ers. Among the 81 microsatellites, seven were EST-SSR

markers and the remaining 74.24% were either failed or

weakly amplified. The percentage of cross-amplification was

25.75%.

This is the first report of microsatellite marker develop-

ment carried out in F. indicus through cross-species amplifi-

cation from related species. Similar successful cross-species

amplification for microsatellites in Penaeids were reported

previously by Xu et al. (1999) and Freitas et al. (2007). The

cross amplification success percentage of microsatellites in

this study was low owing to the mutations in the sequences

flanking microsatellite repeats. The reports of Moore et al.

(1999) gave evidence for a low level of sequence simi-

larity in microsatellite regions among penaeid shrimps. As

per Bezault Etienne et al. (2012), the phylogenetic rela-

tionships and evolutionary distance between the different

groups used for cross amplification from the target species

reflect the success of cross-species amplifications. Likewise,

in the present study, the success percentage was more withthe closely related Fenneropenaeus chinensis (37.8%), then

with Penaeus monodon (34.4%) and least with the distant

species, Litopenaeus vannamei (14.3%). Similar results were

observed in other fish and crustacean species (Gopalakrishnan

et al. 2004, 2006; Jones et al. 2004; Chauhan et al. 2007;

Chen et al. 2012; Huang et al. 2012; Guo et al. 2012;

Kathirvelpandian et al. 2014; Mohitha et al. 2014).

Of the 81 developed microsatellite loci (table 2; 14 loci

from F. chinensis, 35 loci from L. vannamei and 32 from

P. monodon), 47 (58.02%) were perfect, 20 (24.69%) were

compound and the remaining 14 (17.28%) were complex in

nature. Based on Weber (1990), perfect SSRs were the pre-

dominant types of repeats than the compound and complex

types. The present study agrees with this. Among the loci,

Table 2. Summary of microsatellite markers developed in F. indicus.

Total no. of markers screened for cross-priming 396 No. of loci cross-amplified in F. indicus 102Percentage of cross-amplification 25.75% No. of polymorphic microsatellite loci 81 (20.45%) No. of alleles 2–26Average no. of alleles 8.221Molecular weight/allele size range 110–527 bp

Expected heterozygosity ( H e) 0.213–0.938

Type of repeat Number Percentage

Mononucleotide 03 3.70Dinucleotide 51 62.96Trinucleotide 13 16.05Tetranucleotide 12 14.81Pentanucleotide 02 2.47Hexanucleotide 04 4.94

Type of repeat Number Percentage

Simple/perfect 47 58.02Compound 20 24.69Complex 14 17.28



7/8


dinucleotide repeats were dominant (62.96%), as in P. mon-

don (67%) (Xu et al. 1999) and F. chinensis (63.8%) (Wang

et al. 2005; Dong et al. 2006; Gao et al. 2008). Excep-

tionally, trinucleotide repeats were found to be dominant in

Saccharum spp. (Cordeiro et al. 2001).

Trinucleotide (16.05%), tetranucleotide repeats (14.81%),

a few mononucleotide repeats (3.70%), pentanucleotide

repeats (2.47%) and hexanucleotide repeats (4.94%) were

also found (table 2). The relationship between polymor-

phism and the type of SSR motif is still not determined in

penaeids (Wang et al. 2005). Weber (1990), in his study

on the human genome, demonstrated high polymorphism of

trinucleotide and tetranucleotide microsatellite with stable

inheritance. In F. indicus, 16.05% trinucleotide and 14.81%

tetranucleotide repeats were observed with high polymor-

phism and stable amplification. The highest level of polymor-

phism was observed in dinucleotide and trinucleotide perfect

repeat motifs. Development of microsatellite markers with

trinucleotide and tetranucleotide repeats are more valuable

as errors while scoring due to the presence of stutter bandswith dinucleotide repeats is more and can be avoided with

trinucleotide and tetranucleotide repeats (Wang et al. 2005).

The repeat length varied from three (Lvan057, Lvan0511

and PmM16) to 41 (PmMS7HG) with the average length

of 26.08. The tandem repeat sequence of 86.42% of the

microsatellite loci were same as that of the resource species,

while repeat motifs of 13.58% loci differed from that of the

resource species which may be due to the faster repeat evo-

lution without changing the flanking regions, as reported in

fishes by Zardoya et al. (1996).

The level of polymorphism usually expressed as the num-

ber of alleles and the gene diversity (the expected het-

erozygosity). From the cross-species amplification trails,

2–26 alleles were observed in each locus with an average

of 8.221 alleles per locus and the observed heterozygosities

from 0.137 to 0.889. Following the sequential Bonferroni

adjustment, the probability test did not detect any signifi-

cant deviation in allele frequencies from that expected under

( P < 0.001) HWE. None of the loci showed significant link-

age disequilibrium for all pairs of loci ( P > 0.05). It was

therefore assumed that allelic variation at microsatellite loci

could be considered independent. The estimated null allele

frequency was not significant ( P < 0.05) at all tested loci

using different algorithms in MICRO-CHECKER, indicating

the absence of null alleles. The expected heterozygosity of polymorphic loci ranged from 0.213 to 0.938, similar to the

results observed among other shrimps from previous studies

(Zhi-Ying et al. 2006; Gao et al. 2008) indicating the use-

fulness of these markers in population structure studies and

mapping in F. indicus.

The polymorphic microsatellite DNA developed for F.

indicus will provide a valuable resource for commercial

shrimp breeding and selection programmes and genetic stud-

ies including stock identification, diversity assessments, link-

age mapping and parentage analysis in both wild and cultured

stocks of F. indicus.

Acknowledgements

This work was funded by the Department of Biotechnology(DBT), Govt. of India (project sanction order no. BT/PR5772/AAQ/03/241/2005) and DNA sequencing facility was provided byRajiv Gandhi Centre for Biotechnology (RGCB), Thiruvanantha- puram, India. We are grateful to all the Scientists and researchscholars of NBFGR Cochin Centre, especially Dr P. R. Divya,

Dr T. Raja Swaminathan, Dr A. Kathirvelpandian, Ms C. Mohithaand Ms K. B. Sheeba for their help and support; and Dr P. Manoj,RGCB for his support during DNA sequencing.

References

Belkhir K., Borsa P., Goudet J., Chikhi L. and Bonhomme F.1997 GENETIX version 4.02, Genetics logiciel sous windows pour Ia ge ne Tique des populations, available at: http://www.genetix.univ-montp2.fr/genetix/genetix.htm.

Bezault E., Rognon X., Gharbi K., Baroiller J. F. andChevassus B. 2012 Microsatellites cross-species amplificationacross some African cichlids. Int. J. Evol. Biol. 2012 (doi:10.1155/2012/80935).

Chauhan T., Lal K. K., Mohindra V., Singh R. K., Punia P.,Gopalakrishnan A. et al. 2007 Evaluating genetic differentia-tion in wild populations of the Indian major carp, Cirrhinus mri- gala (Hamilton–Buchanan, 1882): evidence from allozyme andmicrosatellite markers. Aquaculture 269, 135–149.

Chen F., Zeng L. and Cheng Q. 2012 Development of thirty-four novel polymorphic microsatellite markers in Coilia ectenes (Clu- peiformes: Engraulidae) and cross-species amplification in twoclosely related taxa. J. Genet. 91, e37–e43.

Cordeiro G. M., Casu R. and McIntyre C. L. 2001 Microsatellitemarkers from sugarcane (Saccharum spp.) ESTs cross transfer-able to Eranthus and Sorghum. Plant Sci. 160, 1115–1123.

Dong S., Kong J., Zhang T., Meng X. and Wang R. 2006 Parentagedetermination of Chinese shrimp Fenneropenaeus chinensis basedon microsatellite DNA markers. Aquaculture 258, 283–288.

Freitas P. D., Jesus C. M. and Galetti P. M. 2007 Isolation and char-acterization of new microsatellite loci in the Pacific white shrimp Litopenaeus vannamei and cross-species amplification in other penaeid species. Mol. Ecol. Notes 7, 324–326.

Gao H., Kong J., Yan B., Yu F., Luan S. and Cai S. 2008 Perma-nent genetic resources: twelve new microsatellite markers for theChinese shrimp Fenneropenaeus chinensis. Mol. Ecol. Resour. 8,325–327.

Gopalakrishnan A., Musammilu K. K., Muneer P. M. A., LalK. K., Kapoor D., Ponniah A. G. et al. 2004 MicrosatelliteDNA markers to assess population structure of red tailed barb,Gonoproktopterus curmuca. Acta Zool. Sin. 50, 686–690.

Gopalakrishnan A., Muneer P. M. A., Musammilu K. K., Lal K.K., Kapoor D. and Mohindra V. 2006 Primers from the ordersOsteoglossiform and Siluriform detect polymorphic microsatel-lite loci in sun-catfish, Horabagrus brachysoma (Teleostei:Bagridae). J. Appl. Ichthyol. 22, 456–458.

Guo X. Z., Zhang G. R., Wei K. J., Guo S. S., Qin J. H. andGardner J. P. 2012 Isolation and characterization of twenty-one polymorphic microsatellite loci from Schizothorax o’connori andcross-species amplification. J. Genet. 92, e60–e64.

Huang W., Liang X. F., Qu C. M., Li J. and Cao L. 2012 Isolationand characterization of twenty-five polymorphic microsatellitemarkers in Siniperca scherzeri Steindachner and cross-speciesamplification. J. Genet. 91, e113–e117.

Jones J. W., Culver M., David V., Struthers J., Johnson N. A., Neves R. J. et al. 2004 Development and characterization of microsatellite loci in the endangered oyster mussel Epioblasmacapsaeformis (Bivalvia: Unionidae). Mol. Ecol. Notes 4, 649–652.


http://www.genetix.univ-montp2.fr/genetix/genetix.htmhttp://www.genetix.univ-montp2.fr/genetix/genetix.htmhttp://www.genetix.univ-montp2.fr/genetix/genetix.htmhttp://dx.doi.org/10.1155/2012/80935http://dx.doi.org/10.1155/2012/80935http://www.genetix.univ-montp2.fr/genetix/genetix.htmhttp://www.genetix.univ-montp2.fr/genetix/genetix.htm


8/8

K. A. Sajeela et al.

Kathirvelpandian A., Gopalakrishnan A., Lakra W. S., Krishna G.,Sharma R., Musammilu K. K. et al. 2014 Microsatellite markersto determine population genetic structure in the golden anchovy,Coilia dussumieri. Biochem. Genet. 52, 296–309.

Miller S. A., Dykes D. D. and Polesky H. F. 1988 A simple saltingout procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.

Mohitha C., Joy L., Divya P. R., Gopalakrishnan A., Basheer

V. S., Koya M. et al. 2014 Characterization of microsatel-lite markers in Silver pomfret, Pampus argenteus (Euphrasen,1788) (Perciformes:Stromateidae) through cross species ampli-fication and population genetic applications. J. Genet. 93, e89– e93.

Moore S. S., Whan V., Davis G., Byrne K., Hetzel D. J. S. andPreston N. 1999 The development and application of geneticmarkers for the Kuruma prawn, Penaeus japonicus and their usein parentage determination and linkage mapping. Aquaculture173, 19–32.

Rousset F. 2008 Genepop’007: a complete reimplementation of thegenepop software for Windows and Linux. Mol. Ecol. Resour. 8,103–106.

Van Oosterhout C., Hutchinson W. F., Wills D. P. M. and ShipleyP. 2004 Micro-checker: software for identifying and correcting

genotyping errors in microsatellite data. Mol. Ecol. Notes 4, 535– 538.

Van Oosterhout C., Weetman D. and Hutchinson W. F. 2006 Esti-mation and adjustment of microsatellite null alleles in nonequi-librium populations. Mol. Ecol. Notes 6, 255–256.

Wang Hongxia., Fuhua Li and Jianhai Xiang 2005 PolymorphicEST–SSR markers and their mode of inheritance in Fennerope-naeus chinensis. Aquaculture 249, 107–114.

Weber J. L. 1990 Informativeness of human (dC–dA)n.(dG–dT)n polymorphisms. Genomics 7, 524–530.

Xu Z., Dhar A. K., Wyrzykowski J. and Alcivar Warren A.1999 Identification of abundant and informative microsatellitesfrom shrimp ( Penaeus monodon) genome. Anim. Genet. 30, 150– 156.

Zardoya R., Vollmer D. M., Craddock C., Streelman J. T., Karl S.and Meyer A. 1996 Evolutionary conservation of microsatelliteflanking regions and their use in resolving the phylogeny of cich-lid fishes (Pisces: Perciformes). Proc. R. Soc. London, B Ser. 263,1589–1598.

Zhi-Ying J. I. A., Xiao-Wen S. U. N., Li-Qun L. I. A. N. G., Da-YuL. I. and Qing-Quan L. E. I. 2006 Isolation and characterizationof microsatellite markers from Pacific white shrimp ( Litopenaeusvannamei). Mol. Ecol. Notes 6, 1282–1284.

Received 24 September 2014, in revised form 21 March 2015; accepted 1 April 2015

Unedited version published online: 9 April 2015

Final version published online: 10 September 2015


fenneropenaeus indicus 81 microsatellite markers

Documents