Polymorphism Identification of Pit1 Gene in Indonesian Buffaloes (Bubalus Bubalis) and Holstein-Friesian Cows

Pit-1 gene has been identified as the pituitary specific transcription factor that regulates the expression of the growth hormone (GH) and prolactin (PRL) genes in the anterior pituitary. The use of polymorphic markers in breeding programmes could make selection more accurate and efficient. A total of 320 Indonesian buffaloes from four populations and 45 FH cows from nine populations were genotyped for polymorphism of Pit1|Hinf1 gene by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) methods. The gene polymorphism was detected only in FH cattle. The frequency of AA, AB, and BB genotype was 2%, 44%, and 53% respectively. The frequency of A allel was 25% and B allel was 75%, but no polymorphism was detected in 320 Indonesia buffaloes.


INTRODUCTION
Indonesian buff aloes (Bubalus bubalis) is one of the important large ruminants to be improved due to its desirable characters to adapt to low quality vegetation and to survive against tropical parasites. A various number of essential functions of buff alo coming from a commonly traditional production system are basically for sources of meat, millq draft power, manure, social and culture a ributes giving a signifi cant contribution to the national livestock production.
In an eff ort to maintain, explore and develop the potential genetic of Indonesian buff aloes, identifi cation of the performance and genetic parameters is needed, primarily related to important economic trait. Origin of information (gene fl ow), the characteristics of livestock (production traits and reproduction) and characteristics of population (diversity, the status of the population) of local buff alo yet well known. Though the information is important for the development and improvement of genetic quality of local buff alo as part of the development of national buff alo, information genetics of Indonesian buff aloes is very low. This might partly be due to the lower performance of buff aloes with respect to economic important traits when compared to ca le, limiting the interests in buff alo genome analysis in the developed countries (Kierstein et al., 2004). However, the domestic buff alo holds a great economic potential in the developing countries. Buff alo is admired as multipurpose animal for dairy, meat, and drought. The stock of domestic water buff alo is estimated to be around 130 million, which is around 1/9 of the total worldwide ca le population (FAO & IINEP, 2000).
Growth and carcass traits, which are under the control of multiple genes, are economically important traits in livestock. Selection of animals with higher growth rate and be er carcass composition is of great signifi cance to breeders and consumers. Current technologies likely enable animal breeder to improve on the accuracy and effi ciency of traditional selection methods by applying genetic markers through marker-assisted selection. Pituitary-specifi c transcription factor responsible for pituitary development and hormone expression in mammals. Pit1, which is also termed growth hormone factor I (GHFI), is a pituitary-specifi c transcription factor that is responsible for pituitary development and hormone expression in mammals. Pit1 is a member of the POU domain containing proteins, which is a group of transcriptional regulators that have a critical role in diff erentiation and proliferation of cells (Zhao, 2002).
The inhibition of Pit1 synthesis has been associated with marked decrease of both GH and PRL expression and proliferation of somatotropic and lactotropic cell lines (Castrillo et al.,l99l).
Bovine Pit1 is a 129 amino acid protein and member of DNA-binding POU family of homeo domain transcription factor. This has been sublocalized to the centromeric region of bovine chromosome 1, located midway between TGLA57 and RM95 (Moody et al., 1995). The RFLP within bovine Pit1 gene with respect to Hinf I restriction enzyme (Pit1/Hinf I) was fi rst identifi ed by Woollard et al. (1994). Several workers since then have investigated its association with growth, body weights, carcass traits, lactating performance (Renaville et al., 1997). Pit1 polymorphism already reported in bovine, sheep, pig and goat. Moody et al. (1995) identifi ed in bovines the A and B alleles of the Pit1 gene polymorphism, which is an exon 6, A to G silent mutation of the bovine Pit1 gene (Curi et al., 2006) which probably is not direct responsible for phenotypic variation, although relationships have been observed between POUlFl polymorphisms and body weight and somatic measures (Renaville et al., 1997;Dybus et al., 2004), weaning and yearling weight.
In goat, Pit1 gene locates in chromosomes lq2l-22 and is separated into six exons containing a POU domain and homeodomain (Woollard et al., 2000). Lan et al. (2007) Yu et al. (1995), reported association of Pit1 polymorphisms with growth and carcass traits in pigs. These results suggest that Pit1 or a closely linked gene to Pit1 may be important in birth weight and carcass fat traits in swine.
Previous studied observed in Iranian river buffalo, indicated low variation of these genes in buff alo (Javanmard et al., 2005). Renaville et al. (1997) showed that there is a high variation of Pit1 gene in FH cows and the A allele (especially the AB genotype) may have an eff ect on milk yield and its components. Therefore, the information genetic variation in pituitary specifi c transcription factors I (Pit1) gene is necessary observe in Indonesian buff aloes and Indonesian Friesian Holstein cows. Therefore, the purpose of this study was to analyze the genetic variability of the Pitl gene in population of the Indonesian buff aloes and FH cows by using PCR-RFLP.

DNA-Extraction
The genomic DNA was isolated using proteinase K digestion followed by Sambrook et al. (1989). Blood samples were washed with destilated water and centrifuged at 8000 rpm for 5 min. The supernatant was discarded. In order to digest protein in the suspension, 400 µl lysis buff er containing proteinase K 10 µl, SDS 40 µl, and 1xSTE 350 µl and the samples were incubated at 55 o C for two hour. An equal volume of phenol-chloroform (1:1 v/v) was added and the two phases were mixed until emulsion was formed. The two phases were separated by centrifugation at 12000 rpm for 5 min. The aqueous supernatant solution was collected in fresh sterilized tubes. Phenol-chloroform extraction was repeated. The DNA was washed with 70% ethanol and dried. Finally the DNA was dissolved with elution buff er.

PCR Condition
The sequences of the forward and reverse primer for amplifi cation of the Pit1 gene was designed based on Javanmard et al. (2005) No acc Y15995 and AM490263 (Table 1). The polymerase chain reaction for the Pit1 gene was performed in a 25 µl reaction mixture, containing 1 µl MgCl 2 , 0.5 µl of each dNTPS, 1 µl of each primer, 10x buff er, I U taq polymerase, 2 µl genomic DNA template and 17.9 µl destilated water. The reaction mixture was placed in a PCR machine with conditions included: an initial denaturation step at 95 o C for 2 min followed by 30 cycles of 95 o C for 45 sec, 60 o C for 1 min, and 72 o C for 1 min and fi nal extension at 72 o C for 3 min.

Genotyping by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism)
Five µl PCR product of Pit1 gene were digested with restriction enzyme Hinfl at 37 o C for at least 14 h. The digested fragments separated by 12% (w/v) polyacrilamid gel using a 100 base pair (bp) molecular weight standard to calculate the size of the amplifi ed and digested fragments were visualized by silver staining and exposed to ultraviolet light. The genotypes of the individual ca le were determined for each polymorphism by analyzing the size of the fragments reported as base pairs.

Statistical Analysis
The allele and genotype frequencies were estimated by direct counting according to Nei procedure (Nei & Kumar, 2000). Polymorphic of exon 6 Pit1 gene was already described by Javanmard et al. (2005) in Iranian buff alo. Sequence analysis of Hinf1 site in the Pit1 gene revealed a mutation at position 1256 that was a G to A (Figure 2). In ca le, polymorphic in intron 5 and exon 6 in Nanyang ca le was described by Xue et al. (2006).

Genetic Polymorphism of the Pit1 Gene in
Indonesian Buff aloes PCR-RFLP (restriction fragment length polymorphism) using Hinf1 restriction enzyme was used to genotype the Indonesian buff aloes. In homozygous animals either a unique band (611 bp, AA variants) or two bands (367 and 244 bp, BB variant). Heterozygous animal gave a three band (611, 367, and 244 bp). No polymorphism was detected in exon 6 Pit1|Hinf1 in four populations of Indonesian buff aloes (Banten, North Sumatera, Central Java, Nusa Tenggara Barat (NTB)). Results on Pit1 polymorphisms by Hinf1 enzyme show just one alleles (B) and all of the genotypes is BB (Figure 3).
In Iranian buff alo (Javanmard et al., 2005), the amplifi cation of the Pit1 gene fragment resulted in a

Genetic Polymorphism of the Pit1 Gene in Indonesian Friesian Holstein Cows
Blood samples from nine populations in Indonesian FH cows were successfully indentifi ed by using PCR-RFLP method. Hinf1 restriction enzyme was used to genotype the Indonesian FH cows. Restriction pa ern of Pit1 Hinf1 gene have three genotypes of AA, AB, and BB upon digestion of the Pit1 HinfI. AA variants showed single band (611 bp), BB variants showed two band (357 and 243 bp), and AB variants showed three bands (611 bp, 367 bp, and 243 bp) (Figure 4).
Similar fi ndings were obtained by many workers who detect the fragments after digestion (Woollard, 1994;Zwierzchowski, 2002;Javanmard et al., 2005). Pit1 polymorphism in bovine and buff alo was detected by Zhao et al. (2004) used SSCP method. In human, Pit1 polymorphism was detected by Kishimoto et al. (2002). Sequence analysis of the polymorphic HinfI site revealed a mutation at position 1256. The mutation was a G to A transition ( Figure 5).
Alel A : 5'----GACAAGCCTAAATCAGAGTTTAT-----3' Alel B : 5 ----GACAAGCCTGAATCAGAGTTTAT-----3' Figure 5. Sequence analysis of the polymorphic HinfI site revealed a mutation at position 1256. The mutation was a G to A transition. Table 2 showed there is no variation genotype in Indonesian buff aloes. Considering the 320 buff alo analyzed, the overall genotype frequencies was BB (100%). The Pit1 B allele frequency was observed to be the highest (100%) in the studied population (Table 1). This result was diff erent to that reported gene frequencies of Pit1 variant in Iranian buff alo (Javanmard et al., 2005). In Iranian buff alo genotype frequencies were 0.567 for AA, 0.4 for AB, and 0.3 For BB. In which, gene frequencies for A allel was 0.766 and B allel was 0.3578.

Genotype and Gene Frequencies of Pit1|Hinf1 in Indonesian Buff aloes
In Iranian buff alo, gene frequency of A allele was higher than that of B allele. But in Indonesia, gene frequency of B allele was higher than A allele. Diff erent result might be caused by diff erent species of buffalo. Javanmard et al. (2005) identifi ed polymorphism of Pit1|Hinf1 in river buff alo, but in this research, identifi ed polymorphism of Pit1|Hinf1 in swamp buff alo. Renaville et al. (1997) reported the association of A allele at Pit1|Hinf1 locus with superior milk and protein yields and inferior for fat percentage in dairy ca le.
Similarly Zwierzchowski et al. (2002) showed that the allele A of the Pit1 locus had positive eff ect on milk production traits. In beef ca le, Zhao et al. (2004) reported that Pit1|Hinf1 polymorphism appears to eff ect growth traits in Angus ca le and may be a candidate gene for Marked Assisted Selection (MAS).
This fact is almost the same with the previous observations of Renaville et al. (1993). The AA pa ern was less frequent than the AB or BB pa erns. The signifi cant superiority of the Pit1 A allele over the B allele was observed for milk yield, protein yield, body depth, angularity, and rearleg set (Jawasreh et al., 2009). Lan et al. (2007)  801 individuals belonging to nine native Bp breeds. PCR product of Pitl gene were digested with Alul restriction enzyme. There were three genotypes, CC, TC and TT. Lan et al. (2007) reported relationships between AluI PCR-RFLPs and eight production traits of 663 samples were done. Signifi cant association of diff erent genotypes with milk yield (P<0.05) and birth weight (P<0.05) implied that Pit1 gene had positive eff ect on milk performance and early weight. While goat Pit1 gene did not signifi cant eff ect on her traits.
Genetic variability (Tables 2 and 3) results in both ca le breeds for the two studied genes indicated the low variation. The high prevalence of B allele and BB genotype suggested almost near fi xation of B allele in Indonesian buff aloes. That may be indicated the specifi c allele in Indonesian buff aloes.

CONCLUSION
No polymorphism found in Pit1|Hinf1 locus in Indonesian buff aloes. The overall genotype frequencies for Pit1 gene was BB (100%). Therefore, Pit1|Hinf1 locus could not be used as a marker for meat production. There was high variation of Pit1 gene in Friesian Holstein cows with genotype frequencies of AA, AB, and BB were 0.02, 0.44, and 0.53 respectively. Gene frequencies of A allele and B allele in Pit1 were 0.25 and 0.75, respectively. Association between Pit1|Hinf1 locus variation with milk production for Friesian Holstein cows could be further investigated.