Genetic Variation of the IGF1 and OPN Genes in Holstein-Friesian Dairy Cattle of Historical and Non-Historical Twins
Genetic Variation of the IGF1 and OPN Genes in Holstein-Friesian
Dairy Cattle of Historical and Non-Historical Twins
Anneke Anggraeni1, *, Hasanatun Hasinah2, Santi Ananda Arta1, Bess
Tiesnamurti2, Restu Misrianti3, & Eryk Andreas4
1)
Indonesian Research Institute for Animal Production,
PO Box 221 Ciawi, Bogor, Indonesia.
*e-mail: [email protected]
2)
Indonesian Centre Research Institute of Animal Sciences,
Jl. Raya Pajajaran, Bogor, Indonesia.
3)
Faculty of Agriculture and Animal Science, Sultan Syarif Kasim Islamic State
University, Riau, Indonesia.
4)
Department of Animal Production and Technology,
Faculty of Animal Science, Bogor Agricultural University, Bogor, Indonesia.
Abstract
The application of DNA molecular techniques can be used to determine the
mutation cases at DNA fragments associated with fertility traits in cattle. This study
was aimed to identify genetic variants of Insulin Like Growth Factor 1 (IGF-1) and
Osteopontin (OPN) genes to be considered as candidate genes controlling fertility
traits in HF cattle of historical twins (27 heads) and non historical twins (15 heads)
from West Java, Indonesia. Historically twinning cattle was defined as either the
cows ever calved twins (more) or their offspring (female and males). Investigation
of genetic variants was done by applying PCR-RFLP method by using restriction
enzyme of SnaB1 for The IGF-1 gene and Bsr1 for the OPN gene. Amplification
produced DNA products of 249 bp (IGF-1 intron-1) and 290 bp (OPN intron-4).
Genotyping on the IGF-1 gene locus Snab1 produced only one DNA fragment,
meaning all the cows having the BB genotype (249 bp). Monomorphic of the IGF1 gene was probably due to no mutation (C/T) at the nucleotide as a cutting site.
Instead, it was discovered genetic variants of the OPN gene locus Bsr1, resulting
three DNA banding patterns, these genotypes were successively CC (200 and 90
bp), CT (290, 200, 90 bp) and TT (290 bp). The proportion of CC, CT and TT
genotypes in historical twinning cattle (30%: 56%: 14%) differed to those of non
historical twinning one (20%: 40%: 40%). Former cattle had the amount of CC and
CT genotypes higher than the latter uterine milk uterus fertility for embryo growth
in HF dairy cows observed.
Keywords: Hostein Friesian, IGF-1 gene, OPN gene, PCR-RFLP, twinning
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
91
Introducton
Productvty of dary cows s strongly determned by the level of ther fertlty. The potency of a cow to gve twn brths needs to be studed to get nformaton
on how far ths trat can be nherted. The nhertance of twn brths n cattle had a
smlar pattern to quanttatve trats, t was controlled by many genes and nteracted
wth envronment (Len et al., 2000). DNA molecular technques focusng on genomc analyss can be used to examne the nsdences of mutaton of the sequences
of DNA fragments related to the changes n breedng values or performances of
valuable trats.
Genetc polymorphsms of two fertlty genes n cattle, ncludng the IGF1 and
OPN genes, usng the method of restrcton fragment length polymorphsms (RFLP)
or others have been studed. The IGF1 gene n cattle s located n the chromosome 5
(BTA5), of whch contanng QTL regons of controllng twn (multple) brths. So
ths gene was possble to be used as a canddate gene to ncrease the genetc potency
of cows to calve twn or multple (Len et al., 2000). The IGF1 gene played an mportant role n regulatng follculogeness and possbly be nvolved also n regulatng multple ovulatons n cows (Km et al., 2009). Therefore, the IGF1 gene could
be used as a postonal canddate gene and ntron 2 IGF1 gene was hghly sgnfcant
(P= 0.003) assocated wth twnnng trats n cattle (Km et al., 2009).
Osteopontn (OPN) gene n cattle s located n the chromosome 6 (BTA6),
closely located to the QTL genes of mlk producton (Leonard et al., 2005). The bovne OPN gene conssted of 6 exons wth the sze of about 7 kb from genomc DNAs
(Gen Bank accesson number: NW_255516) and encoded a 278-AA proten (Kerr et
al., 1991). Regulaton and mmedately functonal mplcaton of the condtons nvolvng the OPN gene gave temporary and partal possbltes as the result of jonng actvtes n developng and ensurng the mantenance of a pregnancy (Johnson
et al., 2003). The objectve of ths study was to determne genetc polymorphsms
of the IGF1 and OPN genes n HF cattle wth hstorcal twn and non-hstorcal twn
rased by small dary farmers n Lembang Dstrct, West Java Provnce.
Materals and Methods
Blood and DNA Samples
A total blood sample number of 42 heads of HF dary cattle consstng 27 heads
of hstorcal twn brth cattle and 15 heads of non-hstorcal twn brth ones as the
control. Genoms DNA was extracted from fresh blood sampes by usng standard
phenol-chloroform protocol (Sambrook and Russel, 2001).
Amplification and Polymorphism Identification
Amplfcaton of IGF-1 and OPN gene fragments was done by usng poly-
92
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
merase chan reacton (PCR) methods. Reagents were used for amplfcaton of both
fragments are 2 µl of DNA sample, 25 pmol of each prmers (Table 1), 200 µM of
dNTPs mxture, 1 mM of MgCl2, and 0.5 unt of DreamTaq™ DNA polymerase wth
ts buffer (Fermentas) n 25 µl of total solutons. Amplfcaton process was runnng
wthn GeneAmp® PCR system 9700 (Appled Bosystems™) wth the condton of
pradenaturaton at 95°C for 5 mnutes, 35 cycles consstng of denaturaton at 95°C
for 30 second, prmer annealng at 60°C for 45 second and extenson at 72°C for 1
mnute, and the fnal extenson at 72°C for 5 mnutes.
Polymorphsm dentfcaton both n IGF-1 and OPN gene fragments were detected by restrcted fragment length polymorphsm (RFLP) methods. The restrcted
enzyme whch used for IGF-1 gen fragment was SnaBI (New England Bolabs) and
for OPN was BsrI wth followng manufacture’s nstructons. The product of RFLP
methods were vsualzed on 2% agarose gel (w/v) whch staned by EtBr (ethdum
bromde). Allele dentfcaton was followed Sadkowska et al. (2006) for IGF-1
gene and Leonard et al. (2005) for OPN gene.
Table 1. Prmers nformaton were used
Gene
Sequence (5’-3’)
IGF-1 F: ATT ACA AAG CTG CCT GCC CC
R: ACC TTA CCC GTA TGA AAG GAA
OPN F: GCA AAT CAG AAG TGT GAT AGA C
R: CCA AGC CAA ACG TAT GAG TT
PCR Restrcton
Product
enzyme
Reference
249 bp
SnaBI
Sadkowska
et al., 2006
290 bp
BsrI
Leonard et
al., 2005
Statistical Analysis
Genotype frequency represents the rato of a genotype to total populaton.
Allele frequency s a rato of an allele to the overall allele at a locus n the populaton.
Mathemathcs model genotype and allele frequency (Ne and Kumar, 2000) s
represented as follows:
Note :
χii
n
nj
N
χi
= iith genotype frequency
= number sample of genotype
= number sample of j genotype
= total sample
= ith allele frequency
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
93
Results and Dscusson
Amplification and Genotyping of the IGF1 Gene Fragment
Amplfcaton of IGF-1 gene fragment was successful to amplfed the 249 bp
fragment were located n ntron 1 of IGF-1 gene. Genetc polymorphsm of the IGF1 gene was detected by PCR-RFLP method by Sadkowska et al. (2006) through the
examnaton of the presence of a C/T base transton at the 472 nucleotde poston
n non-codgn regon of Bos taurus IGF-1 gene. The substtuton of C to T produced
a new SnaBI (IGF-1|SnaBI) restrcton ste. The anmals were genotyped by follow
Sadkowska et al. (2006). Anmal wth homozgot TT was ndcated by the presence
of two fragments .e. 223 and 26 bp, whle the genotype homozgot CC was ndcated by absent of SnaBI restrcton ste and showed only one fragment .e. 249 bp.
The heterozgot CT was ndcated by the presence of three fragments .e. 249, 223
and 26 bp.
The RFPL analyss samples show that the genotype of HF cattle was homozgot
CC. The allele was found are allele C. Ths result caused the frequency of the CC
genotype obtaned are 100%, regardless of the CT and TT genotype are 0%. Ths
result was contrast to those of some prevous studes by detectng the presence of genetc polymorphsms n the bovne IGF-1 gene. Polymorphsm short tandem repeat
(STR) n the 5’flankng regon of ntron 3 on IGF1 gene was dentfed by Krkpatrck (2001). Sngle strand conformaton polymorphsm (SSCP) n the 5’ flankng
regon of ntron 1 on IGF-1 gene was also dentfed as a transton of T/C known as
RFLP|SnaB1 (Ge et al., 2001). Two polymorphsms of the IGF1 gene, the nserton/
dles TTTG (InDel) n ntron 4 and RFLP|DpnI n ntron 5 were found n Norway
cattle (Len et al., 2000).
Amplification and Genotyping of the OPN Gene Fragment
Amplfcaton of the ntron 4 OPN gene whch located at the chromose 6
(BTA6) nvestgated n HF cattle resulted n a fragment length of 290 bp. The amplfcaton product was then restrcted by BsrI enzyme to detect the presence of pont
mutaton n the ntron 4 OPN gene. Genetc polymorphsm of the OPN gene n ths
study followed the methods of Leonard et al. (2005) whch examned the transton
C/T n the 5’ non-code area n the ntron 4 of Bos taurus OPN gene .
The substtuton of C to T produced a new BsrI (OPN|BsrI) restrcton ste.
Anmal wth homozgot CC was ndcated by the presence of two fragments .e.
200 and 90 bp, whle the genotype homozgot TT was ndcated by absent of BsrI
restrcton ste and showed onlyone fragment .e. 290 bp. The heterozgot CT was
ndcated by the presence of three fragments .e. 290, 200 and 90 bp.The analyss
of RFLP on the OPN|SbrI wthn HF cattles wth hstorcal twn from Pangalengan
dstrct show that there was found three genotypes, namely the CC genotype, CT,
and the TT genotype.
94
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
Table 2. Genotype and allele frequency of the OPN gene n HF hstorcal twn and control
Populaton
Cattle (Head)
Pangalengan
Non-twn (0)
Sub total (17)
Twn (10)
Non-twn (15)
Sub total (25)
Twn (27)
Non-twn (15)
Total (42)
Lembang
Total
Genotype Frequency (%)
CC
24 (4)
40 (4)
20 (3)
28 (7)
30 (8)
20 (3)
26 (11)
CT
53 (9)
60 (6)
40 (6)
48 (12)
56 (15)
40 (6)
50 (21)
TT
24 (4)
0 (0)
40 (6)
24 (6)
14 (4)
40 (6)
24 (10)
Allele Frequency (%)
C
50
70
40
52
57
40
51
T
50
30
60
48
43
60
49
Note: (….) was blood samples
The frequences of the occurrences of CC, CT and TT genotypes of the OPN
gene of HF hstorcal twn cattles from Pangalengan dtrct were 24, 52 and 24%
repectvelly. For HF cattles from Lembang dstrct were found some nterestng
thngs. For non-hstorcal twnnng cattles as the controls were dentfed three
genotypes, namely CC, CT and TT genotypes, wth the frequences of the occurences
of the respectve genotypes were succesvely 20, 40 and 40%y. For hstorcal twn
anmals n ths locaton were found none anmal havng the TT genotype (0%), so
those hstorcal twn cattle had only two genotypes of CC (40%) and CT (60%)
respectvely.
Concluson
Genotypng on the ntron 1 regon of IGF-1 gene n the BTA5 n HF cattles
of both hstorcal twn and non-hstorcal twn resulted n no genetc polymorphsm
(monomorphc) as the DNA fragment representng solely the CC genotype. Ths s as
ndcaton of the C/T substtuton n the ntron1 IGF1 gene mght be dsappearence,
so ths gene was unable to be functoned as a canddate gene n studyng twnnng
trats n HF cattles.
Genotypng on the ntron 4 of OPN gene n HF cattles wth hstorcal twns and
non-hstorcal twns resulted n three genetc varance, provdng CC, CT, and TT
genotypes, but ther frequencs was vared. Ths result proved that the C/T transton
n the non-code area on ntron 4 of OPN gene could be used as an early ndcator as
a canddate gene to study ts control on mlk uterus secreton to medate twnnng
brth n n HF cattle.
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
95
References
Ge, W., M.E. Davs, H.C. Hnes, K.M. Irvn andR.C. Smmen. 2001. Assocaton of
a genetc marker wth blood serum nsuln-lke growth factor-I concentraton
and growth trats n Angus cattle. J. Anm. Sc. Vol. 79, No. 7. Page: 17571762.
Johnson, G.A., R.C. Burghardt, F.W. Bazer and T. E. Spencer. 2003. Osteopontn:
Roles n mplantaton and placentaton. Bol. Reprod. 69:1458–1471.
Km, E.S, X. Sh, O. Cobanoglu, K. Wegel, P.J. Berger and B.W. Krkpatrck. 2009.
Refned mappng of twnnng-rate quanttatve trat loc on bovne chromosome 5 and analyss of nsuln-lke growth factor-1 as a postonal canddate
gene. J. Anm. Sc. Vol. 87, No. 3. Page: 835-843.
Kerr, J.M., L.W. Fsher, J.D. Termne and M.F. Young. 1991. The cDNA clonng and
RNA dstrbuton of bovne osteopontn. Gene 108:237–243.
Krkpatrk, B., B.M. Byla and K.E. Gregory. 2001. Mappng quanttatve trat loc
for bovne ovulaton rate. Mamen-Genome 11:136-139.
Leonard, S., H. Khatb, V. Schutzkus, Y.M. Chang dan C. Maltecca. 2005. Effects
of the Osteopontn Gene Varants on Mlk Producton Trats n Dary Cattle. J.
Dary Sc. 88:4083–4086.
Len, S., A. Karlsen, G. Klemetsdal, D. I. Va° ge, I. Olsaker et al., 2000 A prmary
screen of the bovne genome for quanttatve trat loc affectng twnnng rate.
Mamm. Genome 11: 877–882.
Ne, M. and S. Kumar. 2000. Molecular Evoluton and Phylogenetcs. Oxford Unversty Press, New York.
Sambrook, J. and D. Russell. 2001. Molecular Clonng: A Laboratory Manual, 3rd
ed. Cold Sprng Harbor Laboratory Press, Unted State of Amerca.
Sadkowska E, Zwerzchowsk L, Oprzadek J, Strzalkowska N, et al. (2006). Effect
of polymorphsm n IGF-1 gene on producton trats n Polsh Holsten-Fresan
cattle. Anm. Sc. Pap. Rep. Inst. Genet. Anm. Breed. Vol. 24. Page: 225-237.
96
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
Dairy Cattle of Historical and Non-Historical Twins
Anneke Anggraeni1, *, Hasanatun Hasinah2, Santi Ananda Arta1, Bess
Tiesnamurti2, Restu Misrianti3, & Eryk Andreas4
1)
Indonesian Research Institute for Animal Production,
PO Box 221 Ciawi, Bogor, Indonesia.
*e-mail: [email protected]
2)
Indonesian Centre Research Institute of Animal Sciences,
Jl. Raya Pajajaran, Bogor, Indonesia.
3)
Faculty of Agriculture and Animal Science, Sultan Syarif Kasim Islamic State
University, Riau, Indonesia.
4)
Department of Animal Production and Technology,
Faculty of Animal Science, Bogor Agricultural University, Bogor, Indonesia.
Abstract
The application of DNA molecular techniques can be used to determine the
mutation cases at DNA fragments associated with fertility traits in cattle. This study
was aimed to identify genetic variants of Insulin Like Growth Factor 1 (IGF-1) and
Osteopontin (OPN) genes to be considered as candidate genes controlling fertility
traits in HF cattle of historical twins (27 heads) and non historical twins (15 heads)
from West Java, Indonesia. Historically twinning cattle was defined as either the
cows ever calved twins (more) or their offspring (female and males). Investigation
of genetic variants was done by applying PCR-RFLP method by using restriction
enzyme of SnaB1 for The IGF-1 gene and Bsr1 for the OPN gene. Amplification
produced DNA products of 249 bp (IGF-1 intron-1) and 290 bp (OPN intron-4).
Genotyping on the IGF-1 gene locus Snab1 produced only one DNA fragment,
meaning all the cows having the BB genotype (249 bp). Monomorphic of the IGF1 gene was probably due to no mutation (C/T) at the nucleotide as a cutting site.
Instead, it was discovered genetic variants of the OPN gene locus Bsr1, resulting
three DNA banding patterns, these genotypes were successively CC (200 and 90
bp), CT (290, 200, 90 bp) and TT (290 bp). The proportion of CC, CT and TT
genotypes in historical twinning cattle (30%: 56%: 14%) differed to those of non
historical twinning one (20%: 40%: 40%). Former cattle had the amount of CC and
CT genotypes higher than the latter uterine milk uterus fertility for embryo growth
in HF dairy cows observed.
Keywords: Hostein Friesian, IGF-1 gene, OPN gene, PCR-RFLP, twinning
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
91
Introducton
Productvty of dary cows s strongly determned by the level of ther fertlty. The potency of a cow to gve twn brths needs to be studed to get nformaton
on how far ths trat can be nherted. The nhertance of twn brths n cattle had a
smlar pattern to quanttatve trats, t was controlled by many genes and nteracted
wth envronment (Len et al., 2000). DNA molecular technques focusng on genomc analyss can be used to examne the nsdences of mutaton of the sequences
of DNA fragments related to the changes n breedng values or performances of
valuable trats.
Genetc polymorphsms of two fertlty genes n cattle, ncludng the IGF1 and
OPN genes, usng the method of restrcton fragment length polymorphsms (RFLP)
or others have been studed. The IGF1 gene n cattle s located n the chromosome 5
(BTA5), of whch contanng QTL regons of controllng twn (multple) brths. So
ths gene was possble to be used as a canddate gene to ncrease the genetc potency
of cows to calve twn or multple (Len et al., 2000). The IGF1 gene played an mportant role n regulatng follculogeness and possbly be nvolved also n regulatng multple ovulatons n cows (Km et al., 2009). Therefore, the IGF1 gene could
be used as a postonal canddate gene and ntron 2 IGF1 gene was hghly sgnfcant
(P= 0.003) assocated wth twnnng trats n cattle (Km et al., 2009).
Osteopontn (OPN) gene n cattle s located n the chromosome 6 (BTA6),
closely located to the QTL genes of mlk producton (Leonard et al., 2005). The bovne OPN gene conssted of 6 exons wth the sze of about 7 kb from genomc DNAs
(Gen Bank accesson number: NW_255516) and encoded a 278-AA proten (Kerr et
al., 1991). Regulaton and mmedately functonal mplcaton of the condtons nvolvng the OPN gene gave temporary and partal possbltes as the result of jonng actvtes n developng and ensurng the mantenance of a pregnancy (Johnson
et al., 2003). The objectve of ths study was to determne genetc polymorphsms
of the IGF1 and OPN genes n HF cattle wth hstorcal twn and non-hstorcal twn
rased by small dary farmers n Lembang Dstrct, West Java Provnce.
Materals and Methods
Blood and DNA Samples
A total blood sample number of 42 heads of HF dary cattle consstng 27 heads
of hstorcal twn brth cattle and 15 heads of non-hstorcal twn brth ones as the
control. Genoms DNA was extracted from fresh blood sampes by usng standard
phenol-chloroform protocol (Sambrook and Russel, 2001).
Amplification and Polymorphism Identification
Amplfcaton of IGF-1 and OPN gene fragments was done by usng poly-
92
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
merase chan reacton (PCR) methods. Reagents were used for amplfcaton of both
fragments are 2 µl of DNA sample, 25 pmol of each prmers (Table 1), 200 µM of
dNTPs mxture, 1 mM of MgCl2, and 0.5 unt of DreamTaq™ DNA polymerase wth
ts buffer (Fermentas) n 25 µl of total solutons. Amplfcaton process was runnng
wthn GeneAmp® PCR system 9700 (Appled Bosystems™) wth the condton of
pradenaturaton at 95°C for 5 mnutes, 35 cycles consstng of denaturaton at 95°C
for 30 second, prmer annealng at 60°C for 45 second and extenson at 72°C for 1
mnute, and the fnal extenson at 72°C for 5 mnutes.
Polymorphsm dentfcaton both n IGF-1 and OPN gene fragments were detected by restrcted fragment length polymorphsm (RFLP) methods. The restrcted
enzyme whch used for IGF-1 gen fragment was SnaBI (New England Bolabs) and
for OPN was BsrI wth followng manufacture’s nstructons. The product of RFLP
methods were vsualzed on 2% agarose gel (w/v) whch staned by EtBr (ethdum
bromde). Allele dentfcaton was followed Sadkowska et al. (2006) for IGF-1
gene and Leonard et al. (2005) for OPN gene.
Table 1. Prmers nformaton were used
Gene
Sequence (5’-3’)
IGF-1 F: ATT ACA AAG CTG CCT GCC CC
R: ACC TTA CCC GTA TGA AAG GAA
OPN F: GCA AAT CAG AAG TGT GAT AGA C
R: CCA AGC CAA ACG TAT GAG TT
PCR Restrcton
Product
enzyme
Reference
249 bp
SnaBI
Sadkowska
et al., 2006
290 bp
BsrI
Leonard et
al., 2005
Statistical Analysis
Genotype frequency represents the rato of a genotype to total populaton.
Allele frequency s a rato of an allele to the overall allele at a locus n the populaton.
Mathemathcs model genotype and allele frequency (Ne and Kumar, 2000) s
represented as follows:
Note :
χii
n
nj
N
χi
= iith genotype frequency
= number sample of genotype
= number sample of j genotype
= total sample
= ith allele frequency
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
93
Results and Dscusson
Amplification and Genotyping of the IGF1 Gene Fragment
Amplfcaton of IGF-1 gene fragment was successful to amplfed the 249 bp
fragment were located n ntron 1 of IGF-1 gene. Genetc polymorphsm of the IGF1 gene was detected by PCR-RFLP method by Sadkowska et al. (2006) through the
examnaton of the presence of a C/T base transton at the 472 nucleotde poston
n non-codgn regon of Bos taurus IGF-1 gene. The substtuton of C to T produced
a new SnaBI (IGF-1|SnaBI) restrcton ste. The anmals were genotyped by follow
Sadkowska et al. (2006). Anmal wth homozgot TT was ndcated by the presence
of two fragments .e. 223 and 26 bp, whle the genotype homozgot CC was ndcated by absent of SnaBI restrcton ste and showed only one fragment .e. 249 bp.
The heterozgot CT was ndcated by the presence of three fragments .e. 249, 223
and 26 bp.
The RFPL analyss samples show that the genotype of HF cattle was homozgot
CC. The allele was found are allele C. Ths result caused the frequency of the CC
genotype obtaned are 100%, regardless of the CT and TT genotype are 0%. Ths
result was contrast to those of some prevous studes by detectng the presence of genetc polymorphsms n the bovne IGF-1 gene. Polymorphsm short tandem repeat
(STR) n the 5’flankng regon of ntron 3 on IGF1 gene was dentfed by Krkpatrck (2001). Sngle strand conformaton polymorphsm (SSCP) n the 5’ flankng
regon of ntron 1 on IGF-1 gene was also dentfed as a transton of T/C known as
RFLP|SnaB1 (Ge et al., 2001). Two polymorphsms of the IGF1 gene, the nserton/
dles TTTG (InDel) n ntron 4 and RFLP|DpnI n ntron 5 were found n Norway
cattle (Len et al., 2000).
Amplification and Genotyping of the OPN Gene Fragment
Amplfcaton of the ntron 4 OPN gene whch located at the chromose 6
(BTA6) nvestgated n HF cattle resulted n a fragment length of 290 bp. The amplfcaton product was then restrcted by BsrI enzyme to detect the presence of pont
mutaton n the ntron 4 OPN gene. Genetc polymorphsm of the OPN gene n ths
study followed the methods of Leonard et al. (2005) whch examned the transton
C/T n the 5’ non-code area n the ntron 4 of Bos taurus OPN gene .
The substtuton of C to T produced a new BsrI (OPN|BsrI) restrcton ste.
Anmal wth homozgot CC was ndcated by the presence of two fragments .e.
200 and 90 bp, whle the genotype homozgot TT was ndcated by absent of BsrI
restrcton ste and showed onlyone fragment .e. 290 bp. The heterozgot CT was
ndcated by the presence of three fragments .e. 290, 200 and 90 bp.The analyss
of RFLP on the OPN|SbrI wthn HF cattles wth hstorcal twn from Pangalengan
dstrct show that there was found three genotypes, namely the CC genotype, CT,
and the TT genotype.
94
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
Table 2. Genotype and allele frequency of the OPN gene n HF hstorcal twn and control
Populaton
Cattle (Head)
Pangalengan
Non-twn (0)
Sub total (17)
Twn (10)
Non-twn (15)
Sub total (25)
Twn (27)
Non-twn (15)
Total (42)
Lembang
Total
Genotype Frequency (%)
CC
24 (4)
40 (4)
20 (3)
28 (7)
30 (8)
20 (3)
26 (11)
CT
53 (9)
60 (6)
40 (6)
48 (12)
56 (15)
40 (6)
50 (21)
TT
24 (4)
0 (0)
40 (6)
24 (6)
14 (4)
40 (6)
24 (10)
Allele Frequency (%)
C
50
70
40
52
57
40
51
T
50
30
60
48
43
60
49
Note: (….) was blood samples
The frequences of the occurrences of CC, CT and TT genotypes of the OPN
gene of HF hstorcal twn cattles from Pangalengan dtrct were 24, 52 and 24%
repectvelly. For HF cattles from Lembang dstrct were found some nterestng
thngs. For non-hstorcal twnnng cattles as the controls were dentfed three
genotypes, namely CC, CT and TT genotypes, wth the frequences of the occurences
of the respectve genotypes were succesvely 20, 40 and 40%y. For hstorcal twn
anmals n ths locaton were found none anmal havng the TT genotype (0%), so
those hstorcal twn cattle had only two genotypes of CC (40%) and CT (60%)
respectvely.
Concluson
Genotypng on the ntron 1 regon of IGF-1 gene n the BTA5 n HF cattles
of both hstorcal twn and non-hstorcal twn resulted n no genetc polymorphsm
(monomorphc) as the DNA fragment representng solely the CC genotype. Ths s as
ndcaton of the C/T substtuton n the ntron1 IGF1 gene mght be dsappearence,
so ths gene was unable to be functoned as a canddate gene n studyng twnnng
trats n HF cattles.
Genotypng on the ntron 4 of OPN gene n HF cattles wth hstorcal twns and
non-hstorcal twns resulted n three genetc varance, provdng CC, CT, and TT
genotypes, but ther frequencs was vared. Ths result proved that the C/T transton
n the non-code area on ntron 4 of OPN gene could be used as an early ndcator as
a canddate gene to study ts control on mlk uterus secreton to medate twnnng
brth n n HF cattle.
Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
95
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Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012