The polymorphism in g . 1256 G > A of bovine pituitary specific transcription factor-1 ( bPIT-1 ) gene and its association with body weight of Pasundan cattle

Bovine Pituitary specific transcription factor 1 (bPit-1) is one of amino acid that controling pituitary gland in mammals. The pituitary gland is important for secretion of growth hormone from growth genes. This study was carried out to detect polymorphism in the exon 6 of bPit-1 (g.1256G>A) in Pasundan cattle using PCR-RFLP method and its association with body weight. Total of 69 heads (15 males and 54 females) of Pasundan cattle from breeding station (BPPIBT-SP Ciamis, West Java) were used in this study. Research showed that two genotypes of bPit-1/HinfI gene were identified in this study i.e GG (0.90) and AG (0.10) with allele frequencies of 0.05 (A) and 0.95 (G). The polymorphic informative content (PIC) and number of effective allele (ne) values were 0.09 (low) and 1.11. respectively. The Chi-square (χ2) value in the population studied was 0.20 and in Hardy-Weinberg equilibrium (χ2<5.99). It was concluded that the polymorphism of bPit-1/HinfI in Pasundan cattle included of low category and was not associated with body weight.


INTRODUCTION
Pasundan cattle is one of native cattle in Indonesia decided by Ministry of Agriculure No: 1051/Kpts/SR.120/10/2014. Pasundan cattle was created from crossbreding between Bos indicus and Bos javanicus since hundred years ago. This cattle was adapted well in West Java Province and kept by the farmers as beef cattle. Recently, the genetic improvement of Pasundan cattle was supported by local gevernment through breeding station of Balai Pengembangan Perbibitan dan Inseminasi Buatan Ternak -Sapi Potong (BPPIBT-SP) Ciamis, West Java. As the Pasundan breeding center, BPPIBT-SP Ciamis must be capable to increase livestock's productivity through livestock selection. Recently, livestock selection can be conducted based on single nucleotide polymorphism (SNP) in the gene that controling productivity and called as the candidate gene (Dekkers, 2004;Van Eenennaam et al., 2007).
There are many growth hormone family genes that were used as molecular selection in cattle i.e. insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), growth hormone (GH), growth hormone receptor (GHR), growth hormone releasing hormone (GHRH) and pituitary specific transcription factor (Pit-1) genes. The bovine Pit-1 gene is one of the candidate gene that potential for molecular selection in cattle (Sumantri et al., 2011;Oner et al., 2017). The bPit-1 gene was located at centromeric region of chromosome 1 (1q21-22) and consists of five introns and six exons (Woollard et al., 2000). The bPit-1 gene was synthesized at anterior pituitary gland and has 291 amino acid protein (31-33 kDa) with DNA binding POU domain class 1 transcription factor 1 (POUF1) that is responsible for pituitary development and hormone secreting gene expression in mammals, activating expression of growth hormone, prolactin and thyrotropin βsubunit genes (de Mattos et al., 2004).
Identification genotype of bPit-1/HinfI gene in Pasundan cattle is important as the basic information for molecular selection in the future. Despite, the information regarding to bPit-1 gene of Pasundan cattle so far is not reported. The objectives of this study were to identify the polymorphism in the exon 6 of bPit-1 gene and to investigate the influence of genotype type related to body weight in a herd of Pasundan cattle.

Blood Samples and DNA Extraction
A total of 69 heads of Pasundan cattle (15 males and 54 females) from breeding station (BPPIBT-SP Ciamis, West Java Province) were used for blood sampling purpose. Blood samples (3-5 mL) were taken from cocygeal vein using venoject and collected in vaccutainer tubes containing anticoagulant (K2EDTA). The blood samples were used in the DNA extraction kit process using the Genomic DNA Mini kit (Geneaid Biotech Ltd., Taiwan) following the manufactures instruction. The extracted DNA was recorded and stored at -20 o C for next analysis.

PCR Amplification of bPit-1 Gene
The primer sequences for PCR analysis was adoped from Nahavandi et al. (2010) i.e Pit-1F: 5'-GAGCCTACATGAGACAAGCATC-3' and Pit-1R: 5'-AAATGTACAATGTGCCTTCTGA-3'. This primer was amplifed Pit-1 gene along 610 bp according to the reference sequence ( Figure 1). The polymerase chain reaction (PCR) reagents were as follows: 2.7 μL of KAPA2G Robust PCR Kit (Kapa Biosystems, Cape Town, South Africa); each 0.80 μL of forward and reverse primers (200 ng/μL); 2.0 μL of DNA samples; and ddH2O up to 7.0 μL. The PCR was carried out in mastercycler gradient machine (Eppendorf, Germany). The PCR program was set up as follows: initial denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds; annealing at 64°C for 30 seconds; initial extension at 72°C for 30 seconds and final extension at 72°C for 5 minutes. The PCR product was visualized using 1.0% agarose gel (Vivantis, Malaysia). The gel was stained with GelRed TM (Biotium, USA). Total 3.0 μL of 100 bp DNA ladder (Vivantis, Malaysia) was used as molecular size marker. The electrophoresis (110 V; 30 minutes) analysis was used for visualization PCR product with GBOX Documentation System (Syngene, UK).

Genotyping of bPit-1 Gene using RFLP Technique
Analysis of restriction fragment length polymorphism (RFLP) was applied for genotyping of Pit-1 gene in this study. The mixture was consisted of 4.20 μL of PCR product; 0.28 μL of HinfI restriction enzyme (GA*NTC); 0.70 μL buffer and ddH 2 O up to 7.0 μL. Then, the mixtures were incubated at 37 o C for 1 h. Digested products were analyzed using electrophoresis (110 V; 1 h) on 2.0% agarose gel with 3.0 μL of 100 bp DNA ladder. The digested product was stained with GelRed TM and captured with GBOX Documentation System. Samples with AA genotypes were consisted of one DNA fragment (610 bp). Samples with AG genotype consisted of three DNA fragments (610 bp, 367 bp and 243 bp). While, samples with GG genotype consisted of two DNA fragments (367 bp and 243 bp).

Statistical Analysis
Data of body weight (BW) were analyzed applying a linear mixed model as follows: Y i = μ + G i + e i Where: Y i : dependent variable (BW) μ : overall mean G i : fixed effect of the j th genotype (AA, AG, GG) e i : random residual effect The genotype data of in all samples were used to estimate allele frequencies, heterozigosity, polymorphic informative content (PIC), number of effective allele (n e ) and Chi-square (χ 2 ) values as follow: The allele frequencies were calculated using formula from Sadeghi et al. (2008) as follows: Where: X i : frequency of i th allele N ii : number of genotype A i A i N ij : number of genotype A i A j N : number of observation The heterosigosity values were calculated using formula from Nei and Kumar (2000) as follows: Where: H e : expected heterozigosity H o : observed heterozigosity X i : frequency of i th allele X ij : frequency of heterozygote genotype N : number of observation SE : standard error The PIC value was calculated using formula from Hildebrand et al. (1992) as follows: PIC : polymorphic informative content X i : frequency of i th allele X j : frequency of j th allele The n e value was calculated using formula from Nei and Kumar (2000) as follows: Where: n e : number of effective allele X i : frequency of i th allele The χ 2 value was calculated using formula from Nei and Kumar (2000) as follows: Where: χ 2 : Chi-square value O i : number of observed i th genotype E i : numberof expected i th genotype

RESULTS AND DISCUSSION
The Pit-1 gene fragments was successfully amplified using PCR technique for all sample and resulted in a single product of 610 bp (Figure 2). The RFLP analysis showed the fragments obtained for the bPit-1/HinfI polymorphism were 367 and 243 bp for GG genotype; 610, 367 and 243 bp for the AB genotype as presented in Figure  3. The statistical analysis for bPit-1/HinfI polymorphism is presented in Table 1. Genotype AA (610 bp) was not observed in this study and similar to the other breeds cattle such as Golpayegani × Brown Swiss (Javanmard et al., 2005) and Gyr (de Mattos et al., 2004). Despite, Jakaria and Noor (2015) reported that AA genotype in the bPit-1/HinfI gene are absence in many Indonesian native cattle such as Aceh, Katingan and Bali cattle. Therefore, the frequency of AG genotype in this study was 0.10 and similar to Katingan (Jakaria and Noor, 2015). The frequency of A allele in the present study was under 0.10 and similar to native cattle in Indonesia (Madura, Pesisir, Aceh, Katingan, Bali) and Brazil (Gyr) is presented in Table 2.
The PIC value in the present study is low (PIC<0.25) and describes that the genetic diversity of bPit-1/HinfI is not effective for  (Agung et al., 2017). The n e value of bPit-1/HinfI gene in Pasundan cattle was 1.11 and reveals that B allele as the dominant allele in this gene. The genetic diversity of bPit-1/HinfI gene in the animal studied under Hardy-Weinberg (HW) equilibrium and can be caused by random mating still occured in the research site. The H o and H e values in the present study was similar (0.10) and reveal that the animal studied under HW equilibrium. Body weight of Pasundan cattle in GG genotypes was not significantly different from AG genotypes (Table 3). No association between bPit-1/HinfI gene polymorphism and body weight in the present study might be caused by low number of sample. Dybus et al. (2003) reported that in polymorphism of bPit-1/HinfI gene was not associated with body weight in Limousine cattle and similar to the present study. In contrast, Renaville et al. (1997a) reported that A allele in the bPit-1/HinfI gene was found to be superior for milk traits and body measurements in Italian Frieasian Holstein. Morever, Sumantri et al. (2011) reported that genotype AA in the bPit-1/HinfI gene of FH cows had the highest of ovulation rate rather than other genotypes.
The bPit-1/HinfI gene of Pasundan cattle in this study can not be used as molecular selection for body weight. Detection of the polymorphism in the other region of bPit-1 gene i.e. 5'UTR/ promotor, other exons, intron and 3'UTR is important to obtain the genetic marker for productivity traits through marker assisted selection (MAS) program in the future.