Carcass Traits Association with GH/AluI Gene Polymorphism in Indonesian Aceh Cattle

Carcass Traits Association with GH/AluI Gene Polymorphism in
Indonesian Aceh Cattle
Eka Meutia Sari¹,* , Ronny Rachman Noor2, Cece Sumantri2 & Endang Tri
Margawati3
¹ Department of Animal Production, Faculty of Agriculture, Syiah Kuala
University, Banda Aceh, *email: ekasalsa@yahoo.com
2
Department of Animal Production and Technology, Bogor Agriculture University,
Indonesia
3
Research Center for Biotechnology (LIPI), Cibinong, Bogor, Indonesia

Abstract
This study was conducted in order to identify polymorphism of growth hormone
gene in the exon five and to determine the association of GH/AluI polymorphism
with carcass quality in Aceh cattle. A total of 42 DNA genome samples were
extracted from two Aceh cattle population, i.e., Banda Aceh (12), Aceh Besar (30),
and the PCR-RFLP was used to amplify 404 bp of GH gene. The result showed that
the LL genotype was the only genotype found in Aceh cattle population, and the
alele frequency of alel L is 1. This finding indicated that there was not evidence of
polymorphism of GH/AluI in Aceh cattle, and there was not correlation of GH/AluI

gene with carcass quality of Aceh cattle. It could be affected by small number of
sampling size. However, this study suggests that GH gene could be possible used as
genetic marker.
Keywords: Aceh cattle, GH gene, PCR-RFLP, Polymorphism

Introducton
The enhancement of beef cattle’s productvty n Indonesa wll be more
approprate f t s done through a selecton whch s not only based on the phenotype,
but also combned wth drect selecton on the level of DNA whch codes the
phenotype n whch the qualty needs to be mproved. The bovne genome map
made based on markers on genome DNA uses molecular technque such as RFLP,
mcrosatellte, mnsatellte, PCR-RFLP, and PCR-SSCP make t possble to dentfy
gene locus whch are responsble for trat varatons havng economc values.
GH gene as a genetc marker s frequently used n researches because growth
hormone gene (GH) s one of the genes whch nfluence growth (D Staso et al.

104

Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012

2005). Casas et al. (2004) reported that QTL for growth trats, carcass composton
and beef qualty was spread on chromosome 1, 2, 3, 16, 17, 19, 20, 21 and 26. GH
gene has a great role n beef cattle’s performance (Breer. 1999), hence t s very
nterestng to dentfy GH gene polymorphsm on Aceh cattle.

Materals and Methods
Ths research ncluded feld and laboratory actvtes. Feld actvty was conducted n Banda Aceh and Aceh Besar slaughterhouses. The DNA extracton and
characterzaton of GH/Alu I gene dversty was conducted n Anmal Molecular
Genetcs Laboratory, Faculty of Anmal Scence, Bogor Agrculture Unversty,
whereas the examnaton of carcass and meat qualty was carred out n Rumnansa
Besar Laboratory, Department of Anmal Producton and Technology. Ths actvty
was done n October 2009 - November 2010.
Blood Sample of Aceh Cattle
Ths research used Aceh cattle’s meat (muscle) n whch ths cattle s local ones
orgnatng from Aceh provnce. Twelve (12) samples were taken n RPH Banda
Aceh, and thrty (30) samples from RPH Antassalam, Aceh Besar (Table 1). The
sample used was longisimus dorsi muscle on the 12th – 13th rb. The cattle slaughtered
were bred tradtonally, n whch durng daytme, they were herded, and put n stalls
at nghts. The slaughter was done tradtonally.
Table 1 The number of meat sample used for GH/AluI gene analyss

Populaton

Number

RPH Banda Aceh
RPH Aceh Besar
Total

12
30
42

Genome DNA Extraction (Meat)
DNA extracton was done from the meat. The extracton procedure followed
phenol-chloroform method (Sambrook et al. 1989).
GH Gene Amplification
The amplfcaton of GH gene fragment was done usng PCR (polymerase chain
reaction) method. The PCR machne used was thermal cycler (Ependorf 5332). The
arrangement of prmers used can be seen n Table 2.

Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012

105

%;
&%
-:0-
/14

&%
/14
1?->

Tablee 2 Prmers
(;@-8 GH/AluI (Gen Bank M57764.1, Gordon et al.,

1983)

Temperature
Product



$!!
!

Locus
Sequences of Prmers
annealing
$

1D@>-/@5;:
PCR
C-?
0;:1
2>;9
@41
91-@
(41
1D@>-/@5;:
;/10A>1
2;88;C10
;2;>9
91@4;0
'-9.>;;7
GH/AluI
59 ºC
404 bp



F:5’-TAGGGGAGGGTGGAAAATGGA-3’

1:1
9>-:3191:@
;2
59
Genotype
Determination Using PCR-RFLP
A?10
/-:
.1
?11:
5:
(-.81



The genotype determnaton of each ndvdual was done usng restriction
(-.81length

%>591>?

8A 1:
-:7
# 
;>0;:




fragment
polymorphism
(RFLP) whch was vsualzed
on agarose gel 1.5%
wth the buffer of 0,5x TBE (trs borat EDTA), functoned on 100 V for 40 mnutes,
%>;0A/@
and colored wth(19-@A>1
ethdum bromde
on UV trans illuminator. AluI was utlzed as a
";/A?



%&
'1=A1:/1?
;2
%>591>?

cutter 
8A
enzyme for
H
the target gene
fragment.

.< (((
&(((

Data Analysis
Gene Frequency. The genotype
frequency was the rato of the number of a

!%
!!




(41
31:;@E95:-@5;:
;2
1-/4
5:05B50A-8
C-?
0;:1
A?5:3








certan genotype
towards populaton number. Allele frequency was the
rato of

&"%the
C45/4
C-?
B5?A-85F10
;:
-3->;?1
318
 
C5@4
@41
.A221>
;2
a certan
allele towards
whole allele n a locus n a certan populaton. The D
(

.;>-@
(
2A:/@5;:10
;:

)
2;>

95:A@1?
-:0
/;8;>10
C5@4
1@4505A9
.>;9501
;:


frequency of each allele n each locus was counted based on Ne dan Kumar (2000)



C-?
A@585F10
-?
-
/A@@1>
1:FE91
2;>
@41
@->31@
31:1
2>-391:@
formula:
-@X :-8E?5?
= (2n + ∑nj) / (2N)

1:1
>1=A1:/E (41
31:;@E1=A1:/E
C-?
@41
>-@5;
;2
@41
:A9.1>
;2
-
/1>@-5:
31:;@E-@5;
;2
-
/1>@-5:
-88181
@;C->0?
@41
C4;81
-88181
5
8;/A?
5:
-
/1>@-5:



2;>9A8-
*5 

:55  J:56



$

GH Gene Amplification Results



 " 
The"!amplfcaton
of growth hormone (GH) gene fragment done on Aceh




!

"!the poston of ntron 4 and prmers reverse on
cattle showed prmers forward on

(41
-9;C@4
4;>9;:1

31:1
2>-391:@
0;:1
;:
/14
/-@@81
?4;C10
59
the poston of flakng regon 3 (Fgure 1). The GH gene fragment amplfcaton
2;>C->0
;:
@41 ;:

-:0
591>? >1B1>?1
;:
@41
135;:

%5/@
was conducted
usng
cycler
(Ependorf
5332)
machne
on @41>9-8

the temperature

(41


31:1
thermal
2>-391:@

-9 


o
of annealng
63 C.
9-/45:1
;:
@41
@19-@A>1
;2
-::1-85:3

;
:@>;:


.< I

.<

D;:



.135;:

 I

.<



&

.591>?forward
2;>C->0poston,
?
>1B1>?1and
-:0PCR
%&

31:1product
;0A/@
Fgure 1.
prmers reverse
GH gene

106

Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012

(41
>1?A8@

;2
31:1

2>-391:@

-9;?1

318
 

5?
1?1:@10

;:
The result
of gene
fragment
amplfcaton
agarose
gel 1.5%
s
%5/@A>1


(41
81:3@4
;2

31:1
2>-391:@
-9-391:@
-91?A8@
;:
-3->;?1
318
 

Fgure %5/@A>1
2. Vsualzaton
of GH gene fragment amplfcaton result on agarose gel 1.5% (M:
 sample)
>1?1->/4
?-971>

.0;:


 
+-;


 
1




The
amplfcaton results
conducted by Gordon et al. (1983), Yao et al. (1996),
8A


-:0
,-75F-014


 

C5@4
@41
?-91
591>?
?4;C10
@4-@
-::1-85:3
591>?
2;>

?1/;:0?

H
2;>

?1/;:0?
>1?A8@10
5:
Ge et 31:1
2>-391:@
;:

H
al. (2003) and Zakzadeh
et al. (2006)2;>

?1/;:0
-:0
wth the same 
H
prmers
showed that
3;;0

%&

;0A/@

(41

-::1-85:3

@19-@A>1

A?10

5:

@45?

>1?1->/4

C-?



30
?1/;:0
@;
annealng prmers GH/AluI gene fragment on 59 ºC for 80 seconds, 65HºC2;>
for
;.@-5:
;1-0
/81->8E
second, and 57 ºC for 60 seconds resulted n good PCR product. The annealng
temperature
used n ths research was
63 ºC for 45 second to obtan optmal PCR
!!




#!

" 


(41
01@1>95:-@5;:
;2

31:1
31:;@E1?1->/4
C-?
/->>510
;A@
A?5:3
%&&"%
product so that
t can be read clearly.
C5@4
8A
-?
@41
/A@@1>
1:FE91
8A
1:FE91
>1/;3:5F10
G(
/A@@5:3
?5@1
-?10
;:
@41
?1=A1:/1
;2

31:1
$
2>-391:@
.15:3
-91
;.@-5:10
@41E
->1
Identification
of GH gene variants using PCR-RFLP
2>-391:@?
C5@4
@41
81:3@4
;2



-:0

C45/4
->1
7:;C?
-?
81A/E:1
-88181
"
%5/@A>1

The determnaton
of GH gene
genotype

31:1
2>-391:@
-?
9A/4
-?

.1?A8@10
n ths research was carred out usng
(41
/A@@5:3
A?5:3
8A

1:FE91
;:
8A
5:
;:1
75:0
;2
2>-391:@
-
2>-391:@
C45/4
/-:
.1
/A@
@C;
.->?
7:;C:
-?
""
31:;@E1-?
PCR-RFLP wth AluI as the cutter enzyme. AluI enzyme recognzed AG|CT cuttng
@41
2>-391:@
C45/4
/-::;@
.1
/A@
;:1
.->
7:;C:
-?
))
31:;@E-391:@
@4>11
ste. Based
on the sequence of GH gene DNA fragment beng amplfed, two AluI
.->?
7:;C:?
-?
")
31:;@E1?1->/4
%5/@A>1


cuttng stes were obtaned; they are fragments wth the length of 87, 132, and 185
whch are knows as leucyne allele (L) (Pcture 3).
#





















































































The cuttng usng AluI enzyme on AluI GH gene fragment as much as 404 bp
only resulted n one knd of fragment: a fragment whch can be cut (two bars) known

.<
as LL genotype, whereas the fragment whch cannot be cut (one bar) known as VV
genotype and combned fragment (three bars) knowns as LV genotype cannot be
found n ths research (Fgure 3).

.<
The

.<
vsualzaton result usng agarose 1.5% shows that GH/AluI locus on
the
Aceh cattle sample populaton beng observed s unform. The genotype found on

.<


.<
Aceh cattle n ths research s LL genotype. Based on the analyss, the LL genotype
frequency was one (1). Ths made the LV and VV genotype frequency zero (0). Based
on Ne%5/@A>1

)5?A-85F-@5;:
;2

G
dan Kumar (2000) equton, allele
L frequency s 1 and V allele frequency s


31:1
2>-391:@
%&&"%
;:
->-3;?1
318
 
#
#->71>

.1?1->/4
?-91?A8@
A?5:3
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?4;C?
@4-@

8A
8;/A?
;:
@41
/14
/-@@81
?-9/4
5?

nd

Proceeding of the 2 International Seminar on Animal Industry | Jakarta, 5-6 July 2012

107

5:
;:1
75:0
;2
2>-391:@
-
2>-391:@
C45/4
/-:
.1
/A@
@C;
.->?
7:;C:
-?
""
31:;@E1-?
@41
2>-391:@
C45/4
/-::;@
.1
/A@
;:1
.->
7:;C:
-?
))
31:;@E-391:@
@4>11
.->?
7:;C:?
-?
")
31:;@E1?1->/4
%5/@A>1

#






















































































.<


.<


.<

.<



.<

%5/@A>1

)5?A-85F-@5;:
;2

G


31:1
2>-391:@
%&&"%
;:
->-3;?1
318
 
Fgure
3. Vsualzaton of GH|AluI
gene fragment PCR-RFLP on aragose gel 1.5% (M:
#
#->71>

.1?1->/4
?-91?A8@
A?5:3
-3->;?1
 
?4;C?
@4-@

8A
8;/A?
;:
@41
/14
/-@@81
?-9/4
5?

Analysis Results on Aceh Cattle’ Carcass and Meat Quality
The value of parameter average of Aceh cattle carcass and meat qualty can be 
seen on Table 3. The pH value of the meat n ths research has the average of 5.46,
whch shows the pH range of normal meat 5.4 – 5.8. The meat color s n category I
(score 1 – 5) accordng to SNI (Indonesan Natonal Standard, 2008). The degree of
tenderness 4 -5 s consdered moderate, not too tender and not too tough.
Table 3 The results of Aceh cattle’s meat and carcass qualty
n

Average ± standard
devaton

Indonesan Natonal
Standard

42

301.88 ± 125.59

-

Eye muscle area (cm )

42

34.19 ± 3.51

-

pH

42

5.46 ± 0.39

5.4-5.8

Tenderness (kg/cm )

42

4.79 ± 1.74

4-5

Cookng loss (%)

42

35.38 ± 6.24

-

DMA (%)

42

29.94 ± 4.55

-

Meat color

42

3.8 ± 1.81

1-5

Parameter
Weght (kg)
2

2

The research results dentfy that Aceh cattle’s carcass/meat has fner meat
fber and ts color s red. The qualty and meat structure greatly depend on types of
meat and locaton. Marblng s also very nfluenced by the breedng system and food
gven.

Conclusons
Based on ths research results, t can be dentfed that the use of GH/AluI only
resulted n LL genotype and monomorphc, so that t cannot be used as a marker to
108

Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012

assocate wth the carcass qualty on Aceh cattle. Ths phenomenon s lkely due to
the lmted number of samples and the exstence of natural selecton towards LV
and VV genotype as the consequence of Aceh cattle adaptaton’s to the local envronment. Thus, a further research s stll necessary, by usng more samples and f
dversty s found, sequencng needs to be done so that t results n more accurate
research results.

References
Breer TA. 1999. Regulaton of proten and energy metabolsm by the somatotropc
axs. Domest Anim Endocrinol 17:209-218.
Casas E et al. 2004. Identfcaton of quanttatve trat loc for growth and carcass
composton n cattle. Anim Genet 35(1):2-6.
D Staso L et al. 2005. Polymorphsm of the GHR gene n cattle and relatonshp
wth meat producton and qualty. Anim Genet 36:138-140.
Ge et al. 2003. Assocaton of a sngle nucleotde polymorphsms n growth hormone
and growth hormone receptor genes wth blood serum nsuln-lke growth factor I concentraton n Angus cattle. J Anim Sci 81:641-648.
Gordon DF et al. 1983. Nucleotde sequence of the bovne growth hormone chromosomal gene. Moll Cell Endocrinol. 33:81-95.
Sambrook J et al. 1989. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold
Sprng Harbor Laboratory Press, USA.
Yao J et al. 1996. Sequence varatons n the bovne growth hormone gene characterzed by sngle-strand conformaton polymorphsm (SSCP) analyss and ther
assocaton wth mlk producton trats n Holstens. Genetics 144:1809-1816.
Zakzadeh S et al. 2006. Analyss of bovne growth hormone gene polymorphsm n
three Iranan natve breeds and Holsten cattle by RFLP-PCR. Biotechnology
5:385-390.

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