D Balkisson

J Sambrook

JH Ko

Moch Syamsul Arifin Zein

S Sulandari

Sri Sulandari

T Sartika

T Seyama

T Watanabe

Tike Sartika

XY Li

Y Maeda

BMC Proceedings

English

PubMed

Moch Syamsul Zein

Springer - Publisher Connector

Selection of Mx gene genotype as genetic marker for Avian Influenza resistance in Indonesian native chicken

Crossref

PROCEEDINGS Open Access
Selection of Mx gene genotype as genetic marker
for Avian Influenza resistance in Indonesian
native chicken
Tike Sartika1*, Sri Sulandari2†, Moch Syamsul Arifin Zein2†
From International Symposium on Animal Genomics for Animal Health (AGAH 2010)
Paris, France. 31 May – 2 June 2010
Abstract
Background: In previous studies, the Mx Gene has been demonstrated to confer positive anti viral responses in
chicken. The amino acid variation of Asn (allele A) at position 631 was specific to positive antiviral Mx/resistant,
while, that of Ser (allele G) was specific to negative Mx/susceptible. This research was aimed at selecting one of the
native chicken breeds which was found out to be resistant to avian influenza using molecular technique. The
selected breed will then be used as the base population to improve native chicken breed in Indonesia.
Methods: Marker Assisted Selection (MAS) method was used in this research to accelerate the selection process,
since the disease resistance had low heritability value. Polymerase Chain Reaction-Restriction Fragment Length
Polymorphism (PCR-RFLP) technique used to select the genotype of Mx++, Mx+- and Mx– that corresponded to the
positive antiviral activity (Mx++), or those which had positive or negative activity (Mx+-) and negative antiviral
activity (Mx–). There were 200 native hens and 40 cocks used in this experiment. Allele frequency of Mx Gene was
calculated. The productivity indicators such as age at first laying, egg weight and hen weight at first laying and
egg production were also measured. The chicken that had Mx++ and Mx+- genotypes, were selected to produce
offspring.
Results: Result showed that the frequency of the resistant allele (Mx+) was 65% and 60% in laying hens and in
cocks, respectively, while the frequency of the susceptible allele (Mx-) was 35% and 40% in hens and cocks,
resepctively. Age, egg weight and hen weight at first laying and egg production for susceptible genotype were
slightly better than for the resistant genotype which were 172,41 VS 178,81 days; 33,94 VS 32,84 g; 1450 VS 1439 g
and 54,32 VS 48,30 %, respectively.
Background
Indonesia has many varieties of native chicken. Based on
phenotypic performances there are more than 32 dis-
tinctive breeds that are being raised under extensive
and/or intensive systems [1]. Estimated population was
about 230 millions. In Indonesia, meat from native
chicken is more expensive than from commercial hybrid
chicken. The consumers like to pay more as it is tastier
and low fat content. The native chicken eggs are also
more expensive than commercial chicken eggs, because
it can be used as part of traditional herbal drink call
“Jamu”, which is very popular in Indonesia.
Since 2003, Indonesia has outbreaks of Avian Influ-
enza (AI). Naturally, native chicken has ability to resist
the virus controlled by antiviral genes. The Mx proteins
are key components and its coding protein had been
shown to be induced by interferon (IFN) and to inhibit
the replication of RNA virus [2]. Their genetic resistance
was shown to result from the difference in genomic
structure of the Mx gene. Watanabe [3] studied chicken
Mx cDNAs from other breeds to see whether these
* Correspondence: tikesartika@hotmail.com
† Contributed equally
1Indonesian Research Institute for Animal Production (IRIAP), PO BOX 221-
Bogor 16002, Indonesia
Full list of author information is available at the end of the article
Sartika et al. BMC Proceedings 2011, 5(Suppl 4):S37
http://www.biomedcentral.com/1753-6561/5/S4/S37
© 2011 Sartika et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
chickens carried resistant or sensitive character of the
Mx gene to the VSV/vesicular stomatities virus infec-
tion, as compared with the differential antiviral activity
with amino acid substitutions at 15 positions. Only an
amino acid substitution at position 631 was identified to
determine the difference between the antiviral activity of
chicken Mx protein; Asparagine (Asn) corresponded to
the positively antiviral activity and serine (ser) corre-
sponded to the negatively antiviral activity [2-4]. The
chicken Mx protein spans about 2,118 bp, with 13
exons on chromosome 1 of the chicken genome. A total
of 237 single nucleotide polymorphisms were found in
the chicken Mx gene by comparison among 4 directly
sequenced Mx genomic DNA sequences. In this study,
identification of Mx gene by mismatching PCR-RFLP
method can discriminate whether the chicken carry
positive or negative virus activity. Sulandari et al[5]
reported the study of 485 samples from 15 breeds of
Indonesian native chicken by a specific PCR-RFLP tech-
nique showed that the averaged frequency of resistant
allele (A/Mx+ allele) was 62.73% and that of sensitive
allele (G/Mx- allele) was 37.27%. Investigation of distri-
bution of the allele A (Mx+) and G (Mx-) on chickens
has been also reported [2,3,6-9].
The aim of the research was to examine the propor-
tion of allele frequency of Indonesian Native chicken,
especially Kampung chicken at breeding population in
IRIAP (Indonesian Research Institute for Animal Produc-
tion). Selection using resistant Mx gene is effective for
breeding program to increase selected breed as resistant
to RNA virus.
Methods
A total of 240 samples (200 hens and 40 cocks) from one
of the native chickens in Indonesia (Kampung chicken,
selected for egg production for 6 generations) were used
in this study. The fresh blood from chickens was collected
and preserved in 96% absolute alcohol. Genomic DNA
was extracted from whole blood using the phenol-chloro-
form method [10]. PCR-RFLP method was used to geno-
type the G/A SNP at nucleotide position 1,892 in the 13th
exon of coding sequence of the Mx gene using PCR-RFLP
mismatched primers. The mismatch primer sequences [8]
which amplify approximate 100 bp long fragment were as
follows: Forward primer NE-F2 (5’CCTTCAGC
CTGTTTTTCTCCTTTTAGGAA3’) and Reverse primer
NE-R2/R (5’CAGAGGAATCTGATTGCTCAGGCGT
GTA3’) or Reverse primer NE-R2/S (5’CAGAGGAATCT-
GATTGCTCAGGCGAATA3’). The Rsa1 restriction
enzyme was used with a recognition sequence of
5’GT↓AC3’ to cut the fragment at the position of interest
when there is an allele G using primer NE-F2 and NE-R2/
R, while the Ssp1 restriction enzyme was used with a
recognition sequence of 5’AAT↓ATT3’ to cut the
fragment at the position of an allele A using primer NE-F2
and NE-R2/S.
The following PCR condition was used: an initial denature
at 94°C for 5 min, followed by 35 cycles of 60 s at 94°C,
annealing temperature for 60 s at 60°C, and 72°C for 60 s,
and final extension at 72°C for 5 min. PCR product were
analyzed by electrophoresis through 2% agarose gel in 1x
TAE buffer, and stained with ethidium bromide. Ampli-
cons were cleaved with the restriction enzyme the Rsa1
and/or Ssp 1 (1U/µg) for 6-8 hours at 37°C following the
manufacture’s instruction. The digested fragments were
visualized by 12% polyacrylamide gel in constant voltage
160 volt for 4 hours. The gel was stained with silver nitrate
[11] and scanned using Adobe Photoshop.
Allele frequencies were calculated for hens and cocks.
Productivity such as age, egg weight and hen weight at
first laying and egg production based on genotype Mx
gene were analysed using Anova, Minitab V.14.
Results and discussion
Mx gene genotyping
The genomic DNA of 200 hens and 40 cocks were suc-
cessfully amplified. Identification of resistant and sensi-
tive chicken Mx gene was examined by mismatch PCR-
RFLP. The PCR product was cleaved with the restriction
enzyme of the Rsa1 and the digested showing poly-
morphism bands, one band with 100 bp in length (A/A,
homozygous resistant Mx allelic genes); two bands with
100 bp and 73 bp in length (A/G, heterozygous Mx alle-
lic genes); and one band with 73 bp in length (G/G,
homozygous sensitive Mx allelic gene). An example of
genotyping results is presented in Figure 1, and corre-
sponding allele frequency is presented in Table 1.
As shown in Table 1, results from the 240 samples by
specific PCR-RFLP indicate a polymorphism at the Mx
gene (which is putatively associated with AI resistance/
AA
GG  AG
Figure 1 Genotyping of Mx gene by acrylamide gel AA,
Genotype of Mx gene: resistant/resistant AG, Genotype of Mx gene:
resistant/sensitive GG, Genotype of Mx gene: sensitive/sensitive.
Sartika et al. BMC Proceedings 2011, 5(Suppl 4):S37
http://www.biomedcentral.com/1753-6561/5/S4/S37
Page 2 of 4
susceptibility in chicken). The frequency of the resis-
tance allele (A allele) for the hen native chicken was
65%, while for cock native chicken was 60%, and that
for sensitive allele (G allele) was 35% for hen and 40%
for cock native chickens. Investigation of the distribu-
tion of the allele A and G on chickens has also been
reported [2,3,5-8,12]. As reported by Sulandari et al.,
[5],Indonesian native chicken had averaged frequency of
resistant allele (A allele) of 62.73% and sensitive allele
(G allele) of 37.27%. A representative various breed of
Indonesian native chicken such as White Kedu, Golden
Arab, Sentul, Dwarf, Black Kedu, Pelung, Gaok, Kalosi,
Tolaki, Merawang, and Cemani chickens, tend to have a
higher frequency of the resistant allele. Frequency of A
allele in each breed was 0.58, 0.62, 0.63, 0.66, 0.68, 0.69,
0.70, 0.70, 0.74, 0.81, and 0.87 respectively, while Kapas,
Wareng, Nunukan and Silver Arab chickens, as founder
local chicken, had a higher frequency of the sensitive
allele, with a frequency of A allele of 0.32, 0.44, 0.45,
and 0.47 respectively.
Productivity of native chicken
The productivity of native chicken was divided by 3
groups for the Mx genotype presented (Table 2). Statis-
tical analyses showed that for all traits no significant
effect of the genotype. However, descriptively the GG
genotype birds carrying the sensitive allele were rela-
tively slightly better than those carrying the AG/resistant
and sensitive allele and AA genotype/resistant allele.
Age at first laying in GG genotype was lower than the
AG and AA genotype. Similarly, egg weight, hen weight
at first laying and egg production was better in GG
genotype.
Hen day production curve was presented in Figure 2.
Based on genotype Mx gene, hen day peak production
of hen with GG genotype (64%) was slightly better than
of hen with AA and AG genotype (60%). Egg production
of native chicken in this study was better than in com-
mon native chicken (28%) in Indonesia [13]. Overall, the
results indicated that the sensitive allele/ GG genotype
tended to be better in productivity than the resistant
allele/AA genotype.
Conclusion
In conclusion, the frequency of Mx+ gene (A allele) in
Indonesian native chicken was relatively high. The
Table 1 Frequency of allelic hen and cock native chicken
Samples Genotype Frequency of allele
AA/Mx++ AG/Mx+- GG/Mx–
Hens native chicken (200) 80 102 18 f(A) =0,65
f(G) =0,35
Cocks native chicken (40) 15 18 7 f(A) = 0,60
f(G) = 0,40
Table 2 The productivity of hen native chicken for 10
weeks at 1st periods lay
Genotype
AA/Mx++ AG/Mx+- GG/Mx–
Age at first laying
Means (days)
Sdv (days)
CV (%)
Max (days)
Min (days)
178,81
16,15
9,03
221
149
174,28
13,79
7,91
216
152
172,41
15,20
6,97
202
155
Egg weight at first laying
Means (g)
Sdv (g)
CV (%)
Max (g)
Min (g)
32,84
3,54
10,77
46
26
32,85
3,88
11,82
49
26
33,94
4,10
1,88
42
29
Hen weight at first laying
Means (g)
Sdv (g)
CV (%)
Max (g)
Min (g)
1439,11
229,31
15,93
2437
962
1395,26
189,27
13,56
2081
1044
1450,06
231,94
106,39
2023
1197
Egg Production during 10 weeks
Means (eggs)
Sdv (eggs)
CV (%)
Max (eggs)
Min (eggs)
Means (%)
Sdv (%)
CV (%)
Max (%)
Min (%)
37,20
15,11
40,62
69
9
48,30
19,62
40,62
89,61
11,69
39,42
13,75
34,88
69
3
51,19
17,86
34,88
89,61
3,90
41,82
16,36
7,51
66
2
54,32
21,25
9,75
85,71
2,60
EGG PRODUCTION OF NATIVE CHICKEN 
40
50
60
70
80
du
ct
io
n 
(%
)
AA
AG
0
10
20
30
1 2 3 4 5 6 7 8 9 10
weeks
H
D
 p
ro
d
GG
Figure 2 Egg production of native chicken
Sartika et al. BMC Proceedings 2011, 5(Suppl 4):S37
http://www.biomedcentral.com/1753-6561/5/S4/S37
Page 3 of 4
productivity of native chicken measured as age, egg
weight, body weight at first laying and 12 weeks egg
production didn’t differ significantly between Mx
genotypes.
Acknowledgements
This article has been published as part of BMC Proceedings Volume 5
Supplement 4, 2011: Proceedings of the International Symposium on Animal
Genomics for Animal Health (AGAH 2010). The full contents of the
supplement are available online at http://www.biomedcentral.com/1753-
6561/5?issue=S4.
Author details
1Indonesian Research Institute for Animal Production (IRIAP), PO BOX 221-
Bogor 16002, Indonesia. 2Research Centre for Biology, The Indonesian
Institute of Sciences (LIPI), Cibinong 16911, Indonesia.
Competing interests
The author(s) declare that they have no competing interests.
Published: 3 June 2011
References
1. Sartika T, Iskandar S: To be Known Germ Plasm of Indonesian Chicken an
Its Utilization. Indonesian Research Institute for Animal Production (IRIAP),
Bogor-Indonesia;Diwyanto K 2007:.
2. Ko JH, Jin HK, Asano A, Takada A, Ninomiya A, Kida H, Hokiyama H,
Ohara M, Tsuzuki M, Nishibori M, Mizutani M, Watanabe T: Polymorphisms
and the differential antiviral activity of the chicken Mx gene. Genome Res
2002, 12:595-601.
3. Watanabe T: Genetic analysis of antiviral resistant Mx gene in the
chicken. International Workshop on Animal Genome Analysis Zuihou-no-ma,
KKR Hotel Tokyo; 2003.
4. Li XY, Qu LJ, Hou ZC, Yao JF, Xu GY, Yang N: Genomic structure and
diversity of the chicken Mx gene. Poult Sci 2007, 86:786-789.
5. Sulandari S, Zein MSA, Astuti D, Sartika T: Genetic polymorphisms of the
chicken antiviral Mx gene in a variety of Indonesian indigenous chicken
breeds. J Veteriner 2009, 10(2):50-56.
6. Balkisson D, Staines K, McCauley J, Wood J, Young J, Kaufman J, Butter C:
Low frequency of the Mx allele for viral resistance predates recent
intensive selection in domestic chicken. Immunogenetics 2007, 59:687-691.
7. Li XY, Qu LJ, Yaou JF, Yang N: Skewed allele frequencies of an Mx gene
mutation with potential resistance to avian influenza virus in different
chicken population. Poult Sci 2006, 85:1327-1329.
8. Seyama T, Ko JH, Ohe M, Sasaoka N, Okada A, Gomi H, Yoneda A, Ueda J,
Nishibori M, Okamoto S, Maeda Y, Watanabe T: Population research of
genetic polymorphism at amino acid position 631 in chicken Mx protein
with differential antiviral activity. Biochem Genet 2006, 44:437-448.
9. Watanabe T: Polymorphisms of the chicken antiviral Mx gene. Cytogenetic
and Genome Research 2007, 117:370-375.
10. Sambrook J, Fitsch EF, Maniatis T: Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, Cold Spring Harbor Press; 2001.
11. Sulandari S, Zein MSA: Practical Manual of DNA Laboratorium. Research
centre for Biology, The Indonesian Institute of Sciences (LIPI), Cibinong,
Indonesia; 2003.
12. Maeda Y: Polymorphism of Mx gene in Asian indigenous chicken
population. International Symposium of local poultry in Indonesia
Diponegoro University, Central-Java-Indonesia; 2005.
13. Sartika T: Quality improvement of the Kampung chicken by selection
and the investigation on the use of the prolactine promoter gene for
marker assisted selection (MAS) to accelerate selection progress. PhD
thesis Bogor Agricultural University, Animal Science Department; 2005.
doi:10.1186/1753-6561-5-S4-S37
Cite this article as: Sartika et al.: Selection of Mx gene genotype as
genetic marker for Avian Influenza resistance in Indonesian native
chicken. BMC Proceedings 2011 5(Suppl 4):S37.
Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit
Sartika et al. BMC Proceedings 2011, 5(Suppl 4):S37
http://www.biomedcentral.com/1753-6561/5/S4/S37
Page 4 of 4


C: Low frequency of the Mx allele for viral resistance predates recent intensive selection in domestic chicken. Immunogenetics

Genetic analysis of antiviral resistant Mx gene in the chicken.

Genomic structure and diversity of the chicken Mx gene. Poult Sci

Molecular Cloning: A Laboratory Manual.

Polymorphism of Mx gene in Asian indigenous chicken population.

Polymorphisms and the differential antiviral activity of the chicken Mx gene. Genome Res

Polymorphisms of the chicken antiviral Mx gene. Cytogenetic and Genome Research

Quality improvement of the Kampung chicken by selection and the investigation on the use of the prolactine promoter gene for marker assisted selection (MAS) to accelerate selection progress.

Sartika T: Genetic polymorphisms of the chicken antiviral Mx gene in a variety of Indonesian indigenous chicken breeds.

Skewed allele frequencies of an Mx gene mutation with potential resistance to avian influenza virus in different chicken population. Poult Sci

T: Population research of genetic polymorphism at amino acid position 631 in chicken Mx protein with differential antiviral activity. Biochem Genet

To be Known Germ Plasm of Indonesian Chicken an Its Utilization. Indonesian Research Institute for Animal Production (IRIAP), Bogor-Indonesia;Diwyanto K

Zein MSA: Practical Manual of DNA Laboratorium. Research centre for Biology, The Indonesian Institute of Sciences (LIPI),

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3108233

Selection of Mx gene genotype as genetic marker for Avian Influenza resistance in Indonesian native chicken

Abstract

Similar works

Full text

Available Versions

Springer - Publisher Connector

Crossref

Springer - Publisher Connector