Genetic polymorphism of Pit-1 | Hinf I gene in Grati-Ongole Grade cattle at Indonesian Beef Cattle Research Station

The pituitary transcription factor (Pit-1) gene is one of the considered genes that responsible to growth in cattle. A specific Pit-1|HinfI gene polymorphism located within exon 6 (g.1256G>A) has been shown to be associated with growth and milk traits in cattle. This study aimed to identify the Pit-1|Hinf1 gene polymorphism in Grati-Ongole Grade cattle (Grati-OG cattle). A total of 107 genomic DNA of Grati-OG cattle were used in this study. The detection of polymorphism was performed by PCR-RFLP method. Only AB and BB genotypes were succesfully identified with the frequency of 0.009 and 0.991, respectively. Whereas, the frequency of A and B alleles were 0.005 and 0.995, respectively. The observed genotype frequencies in this population do not deviated from HWE. The value of Ho, He and PIC were 0.009, 0.009 and 0.009, respectively. In conclusion, the Pit-1|HinfI gene polymorphism in Grati-OG cattle is monomorphic and hence it should not be used in further association studies.


INTRODUCTION
Growth traits is one of the most important and preferable economic traits for beef production.Most economic traits are controlled by a large number of genes which each contribute a small effect to the trait, the loci responsible for such traits being known as quantitative trait loci (QTL) (Curi et al., 2006).Recently, molecular genetics technologies allow the identification of genetic variation at QTL associated with desirable economic traits in many domesticated animals including in cattle.Furthermore, this technologies could be used to explore the genetic potential of the cattle breeds.Selection to the DNA level of the loci affecting growth traits will be more precise and accurate.
The pituitary transcription factor (Pit-1) is a specific transcription factor in pituitary cells that bound elements to activate the expression of prolactin (PRL), thyrotropin β-subunit and growth hormone (GH) genes (Tuggle and Trenkle, 1996).The Pit-1 encoded by Pit-1 gene or also known as POU1F1 gene.The bovine Pit-1 gene has a length of 18,093 bp, consist of 6 exons and 5 introns (NCBI, 2018) and has been mapped to the centromeric region of bovine chromosome 1 (Moody et al., 1995).Deficient in Pit-1 synthesis decreases in proliferation of cell lines producing PRL and GH (McCormick et al., 1990).A specific Pit-1|HinfI gene polymorphism located within exon 6 (g.1256G>A) has been identified as a silent mutation (Dierkes et al., 1998).This polymorphic site has been widely studied and shown to be associated with growth traits in several beef cattle (Zhao et al., 2004;Xue et al., 2006;Carrijo et al., 2008;Yang et al., 2011) and milk yield and quality in dairy breed cattle (Renaville et al., 1997;de Mattos et al., 2004;Yan et al., 2011).However, there were no association found in several studies (Di Stasio et al., 2002;Dybus et al., 2003;Zhao et al., 2004;Trakovická et al., 2015).This varied results suggest that the Pit-1|HinfI gene polymorphism need to be further investigated in other breeds including Indonesian local cattle.
Ongole Grade cattle (or in Indonesian called as Peranakan Ongole/OG cattle) is one of the most popular Indonesian local breeds cattle.This breed is formed from hybridization result between Ongole cattle (Bos indicus) x Java cattle or Banteng (Bos javanicus) since the "Ongolization Program" in 1915 that imposed by Duth colonial goverment, where the Ongole cattle was imported from Nellore province of India (Hardjosubroto, 1994).The phylogenetic analysis has cluster OG cattle into Bos indicus clade (Hartati et al., 2015).This breed is very popular and has a large contribution in the provision of national meat of Indonesia.The OG cattle breeding programs have been conducted by Indonesian Beef Cattle Research Station (IBCRS), Ministry of Agriculture of Indonesia in Grati since 2004 (socalled as Grati-OG cattle).However, the selection method used is still conventional method and has not used genetic markers.So that, the selection system is has not optimal yet.The use of molecular technology approach is expected to accelerate the production of superior breeding stocks in IBCRS.
Many loci that responsible to growth traits need to be further investigated in OG cattle such as Pit-1 gene.The genetic polymorphism of Pit-1 gene in several Indonesian local beef cattle (such as Bali, Madura, Pesisir, Aceh and Katingan beef cattle) has been reported by Jakaria and Noor (2015).However, it has not been reported in OG cattle.Thus, the aim of this study was to identify the Pit-1|HinfI gene polymorphism in Grati OG cattle.

Animals and DNA Samples
A total of 107 individuals of Grati-OG cattle used in this study were from the Superior Breeding Stock Management Unit (UPBU) at the BCRS, Ministry of Agriculture of Indonesia located in Grati-East Java.Blood samples were collected from jugular vein into 3-mL vaccutainer tubes containing K3E EDTA as anticoagulant.DNA isolation were performed using DNA extraction kit (Qiagen, Taiwan) from whole blood samples and then stored at -20°C for further use.

PCR Amplification
In this study, a pair of primers designed by Moody et al. (1995) were used to amplify of 1301 bp targeted fragments of Pit-1 gene (Pit-1|HinfI) by PCR method.Detailed primers information is given in Table 1.PCR reaction was performed in a total volume of 10 µL containing of approximately 10-12 ng/µL of genomic DNA, 5 µL of MyTaq TM HS Red Mix, 2x (Bioline, USA) , 0.2 µM of each primers and ddH 2 O to a final volume of 10 µL.PCR conditions were as follows : pre-denaturation at 95°C for 1 min; followed by 40 cycles of amplification at 95°C for 15 s, 63.5°C annealing for 15 s and 72°C extension for 10 s; and final extension at 72°C for 5 min.

PCR-RFLP Genotyping
Detection of alleles and genotypes were performed by PCR-RFLP method using HinfI restriction enzyme (Promega, USA).The digestion mixtures and conditions were performed according to manufacturer's instructions.The amplified and digested DNA fragments were separated on 1% and 2% agarose gels, respectively and then stained with GelRed ® 10,000X in water (Biotium, USA).Visualization of fragment band patterns in gels were performed under G-BOX Gel Documentation System (Syngene, UK).The targeted Pit-1|HinfI polymorphism is located at nucleotide position 1256 within exon 6 where G to A (g.1256G>A), according to Dierkes et al. (1998) as presented in Figure 1.

Data Analysis
Polymorphism indexes such as genotype frequency, allele frequency, expected heterozygosity (He) and observed heterozygosity (Ho) were calculated according to Nei and Kumar (2000) and Polymorphism Information Content (PIC) was calculated according to Botstein et al. (1980) by direct counting.The genotypic frequency distribution for its deviation to Hardy-Weinberg Equilibrium (HWE) was analyzed by chi-square test.

RESULTS AND DISCUSSION
Detection of genotypes and alleles in this study were performed by PCR-RFLP method.In this study, the targeted fragment of Pit-1|HinfI gene was succesfully amplified with amplicon size of 1301 bp.The Pit-1 digestion with HinfI endonuclease resulted in three fragments (260, 617 and 424 bp) and four fragments (260, 617, 379 and 45 bp) assigned as A and B alleles, respectively.However, from 107 individuals of Grati-OG cattle studied, only two types of genotype were detected i.e.BB and AB genotypes Pit-1|HinfI Gene Polymorphism in Grati-Ongole Grade Cattle (Hartati et al.) 317   2).The 45 bp fragment is not visible in the gel.The genotype frequencies, allele frequencies and HWE test of Pit-1|HinfI gene in Grati-OG cattle are shown in Table 2.This present study showed that the BB genotype and the B allele were most frequent (0.991 and 0.995), whereas the AA genotype did not detected.The A allele is very rare (0.005) which is only detected from one individual bearing the AB genotype.According to Nei (1987) and Allendorf and Luikart (2007), Such condition of this Pit-1|HinfI gene is stated to be monomorphic.Although, it still at Hardy-Weinberg Equilibrium (HWE).
The genetic indexes such as observed heterozigosity (Ho), expected heterozigosity (He) and polymorphism information content (PIC) in this study showed that Ho, He and PIC value of Pit-1|HinfI gene in Grati-OG cattle population were similar (0.009).This Ho and He value was lower than in other Indonesian local breeds cattle were 0.037 to 0.130 and 0.036 to 0.139, respectively (Jakaria and Noor, 2015).
The polymorphism of Pit-1|HinfI gene in exon 6 (g.1256G>A) has been reported in numerous breeds of Bos taurus, Bos indicus, Bos javanicus and Bos primigenius species and its crossbreeds/hybrids cattle (Table 3).Based on these literatures, it could be understood that the frequency of A allele tends to be higher in Bos taurus and crossbreed/hybrid groups than in Bos indicus, Bos javanicus or Bos primigenius groups.Furthermore, the crossbreeds cattle that are influenced by Bos taurus cattle genetic have increasing proportion of A allele and commonly the frequency exceeds that in Bos taurus groups.This suggests that crossbreeding with Bos taurus cattle may alter the proportion of alleles and increases the genetic diversity in Pit-1|HinfI gene of animals.
The genotypic distribution pattern of Pit-1| HinfI gene found in Grati-OG cattle was tend to    (Mukesh et al., 2008).Furthermore, the B allele is predominant allele in Indonesian local breeds and Bos indicus cattle (Table 3).This similarity pattern may be caused OG cattle as well as Grati-OG cattle are categorized in Bos indicus breeds and descendant of Indian Ongole cattle as supported by the history of "Ongolization Program" (Hardjosubroto, 1994) and molecular evidence, where the Indian Ongole cattle was the anchestor of OG cattle (Hartati et al., 2015).Thus, the high frequency of the B allele in this study confirmed that the B allele was tend to be almost fixed in Zebuine cattle (Bos indicus).Interestingly, it does not occur in Nellore (Curi et al., 2006) which has the common anchestor as OG cattle (Hartati et al., 2015).Likewise in other two Bos indicus breeds, Nanyang (Xue et al., 2006) and Talishi cattle (Nahavandi et al., 2010) in which the frequency of A allele is considerably higher than other Bos indicus cattle.Moreover, when analysis within Indian Bos indicus breeds is categorized based on cattle utility, the frequency of A allele in dairy (0.074) and dual purpose (0.085) were significantly higher than for draft purposes (0.029) (P<0.05)(Mukesh et al., 2008).Grati-OG cattle in Indonesia that is utilized as draft or beef purpose tend to have same allelic distribution with draft-purposed cattle in India in which the A allele is rare.These incidents may be further investigated to know the evolutionary process and the effects of allelic variants based on their utility traits in Bos indicus cattle.The same value between Ho and He indicated as randomly mating within Grati-OG cattle population (Frankham et al., 2002) and this is supported by the HWE test.However, a very low heterozigosity and PIC value indicated that the Pit-1|HinfI gene has a poor diversity and less informative in Grati-PO cattle population.Hence, it should not be used in association studies with growth or any certain traits in the Grati-OG cattle population.Nei (1987) stated that the value of heterozygosity depends on the number of samples, the number of alleles and the frequency of alleles.Decreased heterozygosity could lead to loss of genetic variation in a population and even fixed alleles (Russell, 2010).The strategies to increasing gene diversity of Pit-1|HinfI are through crossing between individuals within and outside the population of Grati-OG cattle or crossbreeding with other breeds to increase the frequency of the favorable allele or genotypes.It should consider the breeding objectives itself.

CONCLUSION
In conclusion, the Pit-1|HinfI gene correspond to g.1256G>A in Grati-Ongole Grade cattle (Bos indicus) is found to be monomorphic and less informative as a genetic marker.Hence, it can not be used in further association analysis between marker and certain phenotypic traits including growth traits.Further investigation of Pit-1|HinfI gene polymorphism in other OG cattle population especially raised by farmers are needed to confirm for its polymorphism status.

Figure 1 .
Figure 1.Schematic Representation of the Position of HinfI Restriction Site and Targeted Polymorphic Site in Amplified Fragment of Pit-1 Gene as Illustrated by Dierkes et al. (1998).The targeted fragments are flanked by a pairs of primer including parts of exon 5, intron 5 and exon 6.The restriction site of HinfI (5' G↓ANTC 3') are shown.In the A allele, nucleotide transition (G>A) at the position of 1256 causing HinfI restriction enzyme do not cut the fragment at this position.Broken lines indicate that exons 5 and 6 extend up-and downstream.

Figure 2 .
Figure 2. Genotype Visualization of Pit-1|HinfI Gene in Grati-PO Cattle in a 2% Agarose Gel.AA = AA genotype ; AB = AB genotype ; M = 100 bp DNA ladder.The 45 bp fragment is not visible in the gel.

Table 1 .
The Information of Primers used to Amplify Pit-1|HinfI Gene in Grati-PO cattle(Moody et al.

Table 3 .
(Jakaria and Noor, 2015)uencies of Pit-1|HinfI Gene in Numerous Cattle Breeds Angus x Qinchuan; 2 Germany Yellow x Qinchuan; 3 Limousine x Qinchuan; 4 Angus x be similar with other Indonesian local breeds cattle such as Bali, Pesisir, Aceh, Madura cattle(Jakaria and Noor, 2015)and especially with 16 Indian local cattle breeds that are all Bos indicus breeds cattle