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: ria.anneke@yahoo.co.id
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 IGF-1 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.
Introducton
Productvty of dary cows s strongly determned by the level of ther fertl-ty. 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
ge-nomc 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
m-portant role n regulatng follculogeness and possbly be nvolved also n
regulat-ng 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
bo-vne 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
n-volvng the OPN gene gave temporary and partal possbltes as the result of jon-ng actvtes n developjon-ng and ensurjon-ng 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
poly-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 de-tected 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
IGF-1 F: ATT ACA AAG CTG CCT GCC CC 249 bp SnaBI Sadkowska et al., 2006 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 = iith genotype frequency
n = number sample of genotype
nj = number sample of j genotype
N = total sample
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 1 gene. Genetc polymorphsm of the
IGF-1 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 Bostaurus 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 nd-cated 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 ge-netc 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 Krkpat-rck (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 ampl-fcaton 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 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.
Table 2. Genotype and allele frequency of the OPN gene n HF hstorcal twn and control
Populaton Cattle (Head) Genotype Frequency (%) Allele Frequency (%)
CC CT TT C T
Pangalengan Non-twn (0) - - -
-Sub total (17) 24 (4) 53 (9) 24 (4) 50 50
Twn (10) 40 (4) 60 (6) 0 (0) 70 30
Lembang Non-twn (15) 20 (3) 40 (6) 40 (6) 40 60 Sub total (25) 28 (7) 48 (12) 24 (6) 52 48
Total Twn (27) 30 (8) 56 (15) 14 (4) 57 43
Non-twn (15) 20 (3) 40 (6) 40 (6) 40 60 Total (42) 26 (11) 50 (21) 24 (10) 51 49
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