Identification of a Single Nucleotide Polymorphism at Hinf-1 Enzyme Restriction Site of Pit-1 Gene on Indonesian Bali Cattle Population

This study aimed to determine the Pit-1|Hinf-1 gene polymorphism in Bali cattle (Bos javanicus) as Indonesian native cattle and besides Madura, Pesisir, Aceh, and Katingan cattle breeds as a comparison. DNA samples were extracted from 488 blood samples consisting of Bali (245 heads), Madura (68 heads), Aceh (25 heads), Pesisir (100 heads) and Katingan (50 heads) cattle. The diversity of the Pit-1|Hinf-1 gene wasere analyzed using PCR-RFLP. Whereas the nucleotide base mutations were identified by sequencing. Genotyping data were analyzed by calculating the allele frequency, observed heterozygosity (Ho) and expected heterozygosity (He) values as well as Hardry-Weinberg equilibrium test using POPGENE 1.31 program. Whereas, Tthe sequence data were analyzed by using MEGA6 program. The Pit-1|Hinf-1 gene fragment analysis showed that Bali, Madura, Pesisir, Aceh, and Katingan cattle had high BB genotype, resulting in B allele frequency of 0.982, 0.963, 0.925, 0.960, and 0.960, respectively. Ho and He values were 0.074-0.130 and 0.036-0.139, respectively. Hardy-Weinberg equilibrium test did not significant for all breed populations, except for Aceh cattle population (P<0.05). Mutation from guanine (G) to adenine (A) was found in Pit-1 gene fragment. Therefore, Pit-1|Hinf-1 gene fragment had low genetic diversity in Bali cattle and other breeds population.

Cellularly, Pit-1 gene has been known as a specific transcription factor controlling expression of growth hormone (GH) and prolactin (PRL) genes in pituitary anterior. Besides, Pit-1 gene is responsively involved in protein and hormone syntheses as well as differentiation and proliferation of pituitary cells (Zhang et al., 2009). In genomic DNA of cattle, Pit-1 resides in 1q21-q22 region of chromosome 1 (Woolard et al., 1994) which is flanked by two microsatellites DNA of TGLA57 and RM95 yielding a total of 129 amino acids consisting of 6 exons distantly spaced by 5 introns (Moody et al., 1995). Restriction fragment length polymorphism (RFLP) provided first evidence of high polymorphism in this gene at Hinf-1 restriction sites, located at 5 and exon 6 (Woolard et al., 1994). The polymorphism, so-called Pit-1|Hinf-1 polymorphism, is indicated by base substitution at the restriction site, in which G is changed to A (Seong et al., 2011). Yet, the change has no effect on the primary structure of transcripted polypeptide (silent mutation).
It is interesting that in beef cattle, Pit-1|Hinf-1 polymorphism significantly affects growth rate of Podolica cattle (Salvagi et al., 2011), Hanwoo cattle (Seong et al., 2011) and Nanyang cattle (Kai et al., 2006). These evidences promoted Pit-1|Hinf-1 polymorphism as a promising gene marker for MAS. However, some reports revealed that there was no correlation between the polymorphism and growth performance as well as their carcass traits of beef cattle (Dybus et al., 2003;Rogerio et al., 2006;Thomas et al., 2007;Gill et al., 2010). Despite abundant works, the importance of Pit-1|Hinf-1 polymorphism was mainly deciphered based on studies on European cattle (Bos taurus) and zebu cattle (Bos indicus).
Yet, comprehensive studies on the status of Pit-1|Hinf-1 polymorphism on Indonesian cattle remain crucial factors on determining the efficiency of this gene as a marker. This work aims to observe diversity and pattern of Pit-1|Hinf-1 polymorphism in Bali cattle, as a model of Indonesian native breed cattle, and compared with other Indonesian crossing cattle breeds through PCR-RFLP methods clarified by DNA sequencing.

Cattle Population, Sampling and Genomic DNA Extraction
To obtain diversity and distribution patters of For genomic DNA extraction, blood samples were drawn randomly from the jugular vein by veterinarians and collected in EDTA containing-9 mL sterilized Venoject glass tubes. Genomic DNA was extracted by using Genomic DNA mini kit (blood and culture cells) (Geneaid, Taiwan) based on the protocol provided by manufacturer. Briefly, the extraction procedures consisted of five steps starting from sample preparation followed by cell lyses and DNA binding. Further steps are washing followed by DNA elution. Concentration and quality of the total genomic DNA were determined by using UV-Vis spectrophotometer. This genomic DNA was further used as a template for PCR amplification.

PCR Ampification, Genotyping, and Sequencing
The DNA fragment of Intron 5 and exon 6 from Pit-1 gene was amplified under polymerase chain reaction (PCR) with two flanking primers as following: 5'-AAA CCA TCA TCT CCC TTC TT-3' and 5'-AAT GTA CAA TGT GCC TTC TGA G-3' for forward and reverse primers, respectively. The expected amplified fragment size was about 451 bp (Wollard et al., 1994) ( Figure   1). For PCR, 25 μL of reaction cocktail was prepared consisting of genomic DNA as a template, 10X buffer, 10 mM dNTP, 50 mM MgCl 2 , 30 pmol of forward and reverse primers, and 2.5 U of Taq polymerase (Promega PCR Core System USA). For the reaction, thermo cycler was set up with the following parameters: denaturation at 94 o C for 60 s, annealing at 60 o C for 45 s followed by For RFLP analysis, digestion reaction using Hinf-I restriction enzyme (G↓ANTC) was prepared. The reaction cocktail consists of 5.0 μL endonuclease free H 2 O, 2.5 μL PCR product, 2.5 μL Hinf-I buffer, and 0.5 μL (5 U) Hinf-I restriction enzyme. The reaction was performed at 37 o C for 16 h or overnight. Digestion product was observed by 2% (w/v) agarose electrophoresis in TBE with 85 V and 200 mA for voltage and current, respectively, for 45 min. For this purpose, agarose gel was prepared with 1X TBE buffer, in which 1 g of agarose was diluted in 50 mL of 1X TBE buffer. Following the electrophoresis, the band on agarose gel was visualized under UV-trans illuminator for genotyping.
Sequencing analysis of intron 5 and exon 6 fragment of Pit-1 gene was performed on the sample showing homozygote (AA, BB) and heterozygote (AB) genotypes using both forward and reverse primers. Accordingly, 19 samples were sent to a sequencing company 1st BASE, Selangor, Malaysia, for sequencing.

Statistical Analysis
Allele frequency, observed heterozygosity (Ho) and expected heterozygosity (He) values, as well as Hardy-Weinberg equilibrium test were calculated by using POPGENE 1.31 software (Yeh et al., 2000). Sequencing result of intron 5 and exon 6 fragment of Pit-1 gene was further analyzed by using MEGA6 program (Tamura et al., 2013).

RESULTS AND DISCUSSION
Amplifcation of Pit-1|Hinf-1 gene fragment from genomic DNA of Bali, Madura, South Pesisir, Aceh and Katingan cattle, performed at 60 o C in its annealing temperature, is shown in Figure 2. Genotyping analysis revealed three types of genotypes, AA, AB, and BB were observed in all cattle. Specifically, BB and AB genotypes were found in Bali, Madura, Katingan and Aceh cattle, meanwhile AA genotype was found only in Pesisir cattle from West Sumatera (Figure 3). The success on gene amplification is certainly affected by annealing temperature, template DNA quality and PCR compounds (Viljoen et al., 2005). In this experiment, the fragment was obtained under annealing temperature of 60 o C which is slightly higher than that of reported by Woollard et al. (1994), which is 54 o C.
It is interesting that genotyping result revealed that AA genotype was only found in one Pesisir cattle. AA genotype was observed as a single band at about 451 bp in 2% agarose gel. Meanwhile, BB genotype was represented by two bands with 244 and 204 bp in their sizes, respectively. Three bands were observed for AB genotype for heterozygote cattle, in which the three bands (451, 244, and 207 bp) were accumulation of unmutated (wild type) and mutated bands. The fragmentation, in term of number and size, of each genotype observed in this experiment is supported by Dybus et al. (2013). Similar result was also reported by Zhang et al. (2009), Han et al. (2010 and Salvagi & Dario (2011) revealing that a single band was observed for AA genotype, meanwhile two-and three bands were observed for BB and AB genotypes, respectively.    Genotyping analysis on Bali, Madura, Pesisir, Aceh and Katingan cattle revealed that allele frequency of Pit-1|Hinf-1 gene fragment was high for B allele, while for A ales was considerably low frequency (Table 2). Table  2 displayed the frequency for B allele was found to be higher compared to that of A allele for all cattle. In addition, there was no significant difference on the allele frequency among the cattle used in this experiment, in which the frequency for Balli, Madura, Pesisir, Aceh and Katingan were 0.982; 0.963; 0.925; 0.960; and 0.950, respectively. This result indicated that B allele is fixed in all cattle used in this experiment (Table 2). Similar result was observed in Zebu cattle (Bos indicus) (Beauchemin et al., 2006;Mukesh et al., 2008) (Table 3). Contradictive result was observed on European beef cattle Bos taurus in which the B allele frequency is decreasing as found in Angus (0.55) and Hereford (0.790) cattle (Moody et al., 1995) as well as Limousine cattle (0.730) (Dybus et al., 2003). Similar evidence of decreasing of B allele frequency was also observed in Bos primigenius cattle, including Podolica cattle (0.700) . Interestingly, as reported by , B allele was found to be low in Zebu cattle (Bos indicus) including Nellore (0.103) and Chanchim (0.117) cattle originated from Brazil (Table 3). Besides, B allele was also found to be low in Brangus (0.181) (Thomas et al., 2007), Hanwoo (0.089) (Han et al., 2010) and cross breed of half-Angus and half-Simmental cross breed (0.000) . The existence of B allele or BB genotype has significant correlation to carcass quality specifically in term of back fat thickness, in Hanwoo cattle (Han et al., 2010), and intramuscular fat in Brangus bulls (Thomas et al., 2007). In Qinchuan cattle, AB genotype was shown to be higher than BB genotype for body  weight and shoulder height (Zhang et al., 2009). Yet, AA genotype was not analyzed due to limited number of samples (Zhang et al., 2009). In addition, there was no correlation between polymorphism in Pit-1|Hinf-1 gene fragment and production traits in Limousine cattle (Dybus et al., 2003).

Observed and expected heterozygocity (Ho and
He, respectively) values indicated that diversities of Indonesian native cattle (Bali cattle) and Indonesian cross-breed cattle (Madura, Pesisir, Aceh, and Katingan) were remarkably low. The values were 0.0370-0.130 and 0.036-0.139 for Ho and He, respectively (Table 4). Other constraints are random matting, the absence of mutation, the absence of selection as well as the absence of migration. Hardy-Weinberg equilibrium status was also found in population of Zebu (Mukesh et al., 2008), red Anatolian (Oztabak et al., 2008), Qinchuan (Zhang et al., 2009), Hanwoo (Han et al., 2010) and Holstein Turkey (Ozdemer, 2012) cattle.
Sequences analysis on A and B allele diversity found in Bali, Madura, Pesisir, Aceh and Katingan cattle revealed that G base was changed to A base at the restriction site of Hinf-I (5-GANTC-3) located in the intron 5 exon 6 fragment of Pit-1 gene (Figure 4). The same mutation (GàA) was also found, and classified as silent mutation, in exon 6 of Pit-1 gene (Seong et al., 2011) which was speculated to have no direct effect on phenotype variation . However, the relation between Pit-1|Hinf-1 polymorphism and carcass quality are significant for Hanwoo cattle (Seong et al., 2011;Han et al., 2010) as well as the relationship between the polymorphism and intramuscular fat, in Brangus bulls (Thomas et al., 2007) or production traits, in Qinchuan cattle (Zhang et al., 2009).
Altogether, this result provides evidence for the use of Pit-1|Hinf-1 fragment in intron 5 and exon 6 as marker candidate for cattle growth (Zhang et al., 2009; and carcass quality (Thomas et al., 2007;Han et al., 2010;Ribeca et al., 2014). Yet, the use of this allele, as well as other desired alleles in Bali cattle specifically and other local breed cattle in Indonesia remain to be formulated to obtain appropriate breeding strategy to maintain and improve the allele frequency or equilibrium in the population.

CONCLUSION
B allele of Bali cattle and other local breed cattles had been found to be low in its diversity. This allele was almost fixed in all cattles and had similar distribution pattern on Pit-1|Hinf-1 gene fragment on intron 5 and exon 6. Polymorphism in this fragment was indicated by the change of G base to A base.  genotypes. The arrow indicated the base position that changes (G base to A base) due to polymorphism. R indicates either G or A base.