MA Mu’in and S Lumatauw/Animal Production 15(1):1-7, January 2013

Identification of MspI Polymorphism in the Forth Intron of Chicken Growth

Hormone Gene and Their Associations with Growth Traits
in Indonesia Native Chickens
MA Mu’in* and S Lumatauw

Faculty of Animal Science, Fishery and Marine Science, Papua State University
Jl. Gunung Salju, Amban, Manokwari 98314, West Papua, Indonesia
*Corresponding author email: muinunipa@gmail.com

Abstract. The objective of this research was to identify MspI polymorphism in the forth intron of chicken
growth hormone (cGH) gene and their associations with growth traits in native Indonesian chickens. A total of
72 Indonesia native chickens were genotyped for a locus in the forth intron of cGH gene (cGH-I4/MspI locus)
by PCR-RFLP with MspI restriction enzyme. The result showed two genotypes in this locus: AA and BB, with the
frequency of 90.28% and 9.72%, respectively. Based on body weight average, B allele had a beneficial effect in
increasing the live body weight. The result of General Linier Models analysis indicated that the polymorphism
of this locus had significant association (P<0.05) with body weight at 4 months of age and so did the daily gain
between 2 to 4 months of age. Therefore, these results suggest that there is a possibility of cGH-I4/MspI locus
acting as a genetic marker for growth traits of native Indonesian chickens, especially for body weight at 4
months and daily gain between 2 to 4 months of age.

Keywords: polymorphism, cGH gene, growth traits, Indonesia native chickens.

Abstrak. Tujuan dari penelitian ini adalah untuk mengidentifikasi polimorfisme MspI intron ke-4 pada gen
hormon pertumbuhan ayam (cGH) dan hubungannya dengan sifat pertumbuhan pada ayam asli Indonesia.
Sebanyak 72 ekor ayam asli Indonesia diidentifikasi gentotipenya untuk lokus pada intron ke-4 dari gen cGH
(cGH-I4/MspI lokus) dengan PCR-RFLP mengguakan enzim restriksi MspI. Hasil penelitian menunjukkan adanya
dua genotipe pada lokus ini: AA dan BB , dengan frekuensi masing-masing 90.28 % dan 9,72 %. Berdasarkan
rata-rata bobot badan, alel B memiliki pengaruh yang menguntungkan dalam meningkatkan bobot badan
hidup. Hasil analisis General Linier Model menunjukkan bahwa polimorfisme lokus ini memiliki hubungan yang
nyata (P<0,05 ) dengan bobot badan pada umur 4 bulan dan begitu pula dengan pertambahan bobot badan
harian antara umur 2 sampai 4 bulan. Oleh karena itu, hasil ini menunjukkan bahwa ada kemungkinan cGH-
I4/MspI lokus bertindak sebagai penanda genetik untuk sifat pertumbuhan ayam asli Indonesia, terutama
untuk bobot badan pada umur 4 bulan dan pertambahan bobot badan harian ayam antara umur 2 sampai 4
bulan.

Kata kunci: polimorfisme , gen cGH , sifat pertumbuhan , ayam asli Indonesia.

Introduction economic traits of animals, therefore that

polymorphic loci can be used as genetic
Currently, technology of molecular biology markers for the trait. Furthermore, that specific
has been used in many fields, including farm loci becomes useful as a selection criterion for
animals to determine chicken productivity genetic improvement of these properties.
improvement through a molecular approach. The chickens growth hormone (cGH) is a
Gene mapping has resulted many discoveries of polypeptide hormone, consisting of 216 amino
polymorphic loci in the chicken’s genome. acids (Tanaka et al., 1992). The cGH synthesized
Single nucleotide polymorphism (SNP) in the in and secreted by pituitary gland, is involved in
chicken genom has been identified with a a variety of physiological functions such as
frequency of 1 SNP per 225 bp, which is 5 times growth, body composition, egg production,
in humans (Vignal et al., 2002). When a ageing and reproduction (Stephen et al., 2001;
polymorphic locus is associated with the Wardecka et al., 2005; Thakur et al., 2006;

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 MA Mu’in and S Lumatauw/Animal Production 15(1):1-7, January 2013

Jafari et al., 2009). The cGH gene located on Materials and Methods
chromosome number 19 in avian (Stephen et
Data on body weight and DNA samples.
al., 2001), and contains 5 exons and 4 introns
Data of body weight at 1, 2, 3, and 4 months of
with an overall length of 4.1 kb (Kansaku et al.,
age in 72 chickens and each of its genome DNA
2008).
were used as materials in this study. Both of
Some studies have found polymorphisms
these materials were obtained from previous
along the cGH gene and several polymorphisms
studies (Mu'in et al., 2009). In this study,
correlated with the growth traits in some
genomic DNA were isolated from blood
breeds of chickens (Stephen et al., 2001;
samples of native Indonesian chickens using
Bingxue et al., 2003). Zhang et al. (2007) also
phenol-chloroform extraction method
found that AvaI polymorphism in intron 3 of the
(Sambrook et al., 1989). The genomic DNA
cGH gene in the chickens was significant
samples were stored at -700C. DNA analysis was
associations with abdominal fat weight and
conducted at the Centre for Biotechnology,
abdominal fat percentage. Kuhnlein et al.
Gadjah Mada University, Yogyakarta from
(1997) analyzed 12 strains of non-inbred White
August to September 2010.
Leghorn chickens using PCR-RFLP MspI in intron
1, 3 and 4, and SacI PCR-RFLP in intron 4, DNA amplification. Amplification of specific
demonstrating that alleles in the intron were DNA fragments conducted in intron 4, size 713
associated with egg production traits. Bingxue bp, spanning from the 2479 base to 3192 of the
et al. (2003) found a MspI polymorphism in chicken cGH gene (GenBank: D10484.1). The
intron 4 in the cross breed of chicken (broiler amplification process was performed using a
Star x Silky) associated with breast muscle pair of primers (forward: 5'-cgc-tct-gct-att-tct-
weight. ctt-ac-3' and reverse: 5'-atg-gaa-ccg-tgg-aat-
Screening results of cGH genes from four gat-gg-3'). A total of 19 µl dH2O, 2 µl of DNA
groups of chickens found 46 single nucleotide solution (± 50 ng) and a pair of specific primers,
polymorphisms (SNPs). Four of them (4 SNP) each with 2 µl (16 pmol) were inserted into a
were found in the 5'UTR (untranslated region), 0.2 ml tube of Ready-To-Go PCR Beads
1 SNP in the 3'UTR, 5 SNPs in exon, and 36 SNPs (Amersham Biosciences), mixed until
the intron regions (Nie et al., 2002; 2005). It is homogeneous, then inserted into the PCR
also informed that some of them found to be machine. PCR conditions was programmed as
significantly correlated with body weight, shank follows: initial denaturation 94oC for 5 min, 30
length and weight gain (0 to 4 months). cycles with each cycle, denaturation 94oC for 60
More recently, Lumatauw and Mu'in (2011) sec, annealing 60°C 60 sec, extension 72oC for
revealed non-significant (P>0.05) association of 60 sec, and final extention 72oC for 10 min
polymorphism locus in the intron 3 of cGH (Bingxue et al., 2003).
genes (cGH-I3/EcoRV locus) with body weight at PCR product digestion. PCR products
age 1, 2, 3 and 4 months in native Indonesian (amplicons) obtained were digested with MspI
chickens. This study aimed to detect single restriction enzyme (sequence: c|cgg). A total of
nucleotide polymorphisms in intron 4 of the 10 µl PCR product, 1 µl restriction MspI
cGH gene (cGH-I4/MspI locus) using PCR-RFLP enzyme, 1.5 µl buffer and 2.5 µl aquabides
and its association with growth traits in native (dH2O) were put in a 1.5 ml micro tube, and
Indonesian chickens. incubated at 37°C temperature for 2 hours.

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The digests with the MspI enzyme were weight age or weight gain during the period
electrophoresed through 1.5% agarose gel observation of a certain age were observed in
(contain GoldView Nucleic Acid Stain) in TBE this study on the type of sex (S) the ith genotype
buffer. The procedure: 5 µl digestion was mixed (G) to jth, and kth replicates; μ = general mean;
with 2 µl product loading buffer, and then Si = effect of the ith sex; GJ = effect of the jth
inserted into the gel wells. The running gel was genotype; εijk = random error of sex (S) the ith
performed at 100 volts for ± 30 minutes. The genotype (G) to-j, and the kth test.
electrophoresed gels were vizualized under
ultraviolet light and photographed. Results and Discussion
Genotypes identification. The Identification MspI polymorphism. Detection of single
of genotypes procedure used in this study was nucleotide polymorphisms in intron 4 in the
adopted from Tanaka et al., 1992 as follows: AA chicken GH gene studied had been successfully
genotypes were those shown by a DNA performed using the PCR-RFLP/MspI technique.
fragment (size 713 bp), genotypes AB were In this study, the target DNA fragment size of
shown by the three DNA fragments (sizes 713, 713 bp was located in intron 4 of the chicken
519 and 194 bp), and BB genotypes were GH gene, extending from the base 2479 to 3192
shown by two DNA fragments (sizes 519 and of the chicken GH gene (Figure 1). Previous
194 bp). studies indicated this DNA fragment contained
Statistical analysis. Genotypic and allelic mutation point. The mutation was caused by a
frequencies of cGH-I4/MspI locus were substitution of cytosine nucleotide (c) with
calculated according to the formula of Nei and thymine nucleotide (t) on the 2998 base
Kumar (2000), below: sequences of the chicken GH gene that could
be detected by using MspI (Bingxue et al.,
Xii = (nii/N) x 100% 2003).
Xi = (2nii + ∑nij)/2N Amplification of specific DNA fragment (713
i≠j
bp) was successfully performed using the
where:
primer pair, ie forward: 5'-gtccgtgctcttctcttatc-3
Xii = iith genotype frequency; nii = number sam-
'and reverse: 5'-gcgcaggcttccatcagtat-3' in PCR
ple of ii genotype; nij = number sample of ij
conditions as recommended by Bingxue et al.
genotype; N = total sample; Xi= ith allele
(2003) . The results of electrophoresis of PCR
frequency. If allele frequencies of the locus
products obtained in this study (Figure 2)
studied were not exceed 0.99 (Harris, 1994)
appeared as a clear single band, and occupied
means that locus was categorized polymorphic.
the appropriate position (sized 713 bp).
The associations between the MspI
Digestion with MspI to the PCR product (713
genotypes and the growth traits were analyzed
bp) obtained in this study resulted in two kinds
using General Linier Models (GLM) from
of alleles, B allele (the two bands, 519 and 194
MINITAB Release 13.20 for Windows. Analysis
bp size ) and A allele (single band sized 713 bp,
of the relationship between the polymorphic
and its position parallel to the PCR product). B
loci (GH-I4/MspI) with the chicken growth traits
allele of cGH-I4/MspI locus was demonstrated
(weight age of 1, 2, 3, and 4 months), and the
by the success of the MspI (5 '-c↓cgg-3') found
body weight gain at the age of 1-2 months, the
DNA sequences that were recognized
age of 1-3 months, age 1 - 4 months, age 2-3
throughout the PCR products and success to cut
months, the age of 2-4 months and the age of
them into two fragments sized 519 bp and 194
3-4 months), were studied through the model:
bp. In contrast, the A allele is indicated by the
Yijk = μ + Si + GJ + εijk where: Yijk = specific

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failure of MspI find a recognizable DNA by Tanaka et al. (1992) and Bingxue et al.
sequences along the PCR products. As a result (2003).
the size of the PCR products before and after These results indicate that the frequency of
digested remain the same, which is 713 bp. The alleles A and B of the GH-I4/MspI loci studied in
MspI failed to find DNA sequences that were chicken were 0.9028 and 0.0972 respectivelly.
recognized due to the-2998 nucleotide This means that the A allele is a common allele.
sequence (Figure 3) occupied by thymine Thus the GH-I4/MspI locus in chicken can be
nucleotide (t). The results were consistent with categorized as polymorphic loci, because the
the previous research that had been reported most common A allele frequency is not

Note: E-1: Exon-1; I-1: Intron-1; E-2: Exon-2; I-2: Intron-2; E-3: Exon-3; I-3: Intron-3; E-4: Exon-4; I-4: Intron-4; E-5: Exon-5.
ccgg: sekuen MspI, restriction site: 5’-...c|cgg...-3’
cgctctgctatttctcttac: primer forward; ccatcattccacggttccat: primer reverse

Figure 1. The chickens growth hormone gene (exons and introns) and the sequence of DNA
fragment in the forth intron of chicken growth hormone (size: 713 bp) extending from 2479 to 3192
nucleotides of chicken growth hormone (source: GenBank No.D10484.1) and restriction site at
position 2998 (c to t).

Figure 2. PCR product in the forth intron of Indonesia native chicken growth hormone gene analysed
in a 1.5% agarose gel containing GoldView Nucleic Acid Stain. Lane 1: DNA marker, lanes 2 – 13: PCR
product (713 bp)

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Figure 3. MspI restriction pattern of cGH-I4 primer (forward and reverse primer) using 713 bp PCR
product illustrated in Fig 1.

exceed 0.99 (Harris, 1994). Pipalia et al. (2003) results of the analysis as presented in Table 1 in
found that A allele also showed higher this study showed that the average body weight
frequency (0.8878) in Bantamised White and weight gain of BB genotype is higher than
Leghorn, while the allele frequencies of A dan B of the AA genotype. Significant differences in
alleles were found intermediate in Bantam body weight (P <0.05) was found in chicken at
genetic groups, and in White Leghorn group the age of 4 months while the significant
showed lack of polymorphism with only A allele differences in weight gain was found only in the
present. period of 2 to 4 months. In contrast, Pipalia et
MspI genotypes found in this study were AA al. (2003) reported that non-significant effect of
and BB, whereas AB genotype was not found. genotypes at this locus on body weight 8, 20,
AA genotype was found more (65 chickens as and 40 weeks of age in Bantam and Bantamised
compared to BB genotype (only 7 chickens). White Leghorn.
This is in contrast with the research results
done by Bingxue et al. (2003) reporting three
genotypes: AA (253 chickens), AB (71 chickens),
and BB (9 chicken) in a population of hybrid
chickens (broilers Star x Silky). These studies,
however demonstrated that the AA genotype is
a genotype commonly found in poultry
populations. Figure 4 shows the genotypes of
the GH-I4/MspI locus were found in this study.

Association of MspI polymorphism with the

growth traits. Table 1 shows that the average
body weight and the body weight gain of BB
Figure 4. The PCR-RFLP electrophoresis product
genotypes were higher than of the AA genotype
of chickens growth hormone locus (cGH-
at any age or period of observation. These I4/MspI locus) in Indonesia native chickens
indicates that the B allele has a positive effect using MspI enzyme. Lane 1: DNA markers (M),
on body weight and weight gain of Indonesia lane 2: PCR product, P (713 bp); lanes 3 dan 4:
native chickens until the age of 4 months. The BB genotype (519 dan 194 bp); lanes 5 and 6:
AA genotype (713 bp).

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Table 1. Average body weight and weight gain (g) of Indonesia native chickens aged 1, 2, 3,
and 4 months by sex and genotypes of the cGH-I4/MspI locus
Genotypes
Traits AA BB P
(n=65) (n=7)
a a
Body weight (1 month) 322.1± 61.3 354.3± 52.6 0.202
a a
Body weight (2 months) 646.0±166.9 721.4±139.7 0.257
a a
Body weight (3 months) 1038.5±311.7 1247.1±260.7 0.063
a b
Body weight (4 months) 1474.9±452.1 1793.0±365.0 0.047
a a
Weight gain (1-2 months) 323.8±130.7 367.1±122.2 0.439
a a
Weight gain (1-3 months) 761.3±284.4 892.9±259.0 0.092
a a
Weight gain (1-4 months) 1152.8±423.0 1439.0±366.0 0.060
a a
Weight gain (2-3 months) 392.5±183.6 525.7±227.0 0.061
a b
Weight gain (2-4 months) 828.9±331.4 1071.0±300.0 0.045
a a
Weight gain (3-4 months) 436.5±187.6 545.7±138.7 0.133
P = probability; Different superscripts in the same row indicate significant differences (P <0.05).

Shahnaz et al. (2008) also found that frequency Acknowledgement

of RFLP patterns in this locus did not differ
significantly for body weight at 20 and 36 weeks This research was funded by the grant
of age in three breed groups (Bantam, provided by of DP2M DIKTI, Ministry of
Bantamised White Leghorn, and White Leghorn Education and Culture, Indonesia through
birds). Instead of the body weight and weight “DIPA Universitas Negeri Papua” (Contract: No:
gain, other researchers found that the B allele is 247/H42/KU/2010, April 26, 2010).
positively associated with breast meat weight.
The presence of B alleles in a genotype is more
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