Growth Hormone Gene Genotyping by Msp I Restriction Enzyme and Pcr-rflp Methods in Aceh Cattle Breed at Indrapuri District of Aceh Province

The objective of this research was to identify growth hormone (GH) genes genotype in selectedAceh cattle at Indrapuri's Breeding and Forage Centre (IBFC) of Aceh Cattle. Fourty one cattleconsisting of 21 male and 20 female cattle were used in this study. The genomic DNA was extractedfrom blood using Sambrook et al. (1989) methods. Polymerase Chain Reaction - Restriction FragmentLength Polymorphism (PCR-RFLP) and mehod of sequencing was used to detect MspI site on GH gene.Based on sequencing results, it can be concluded that all cattle were monomorphism. The frequency ofTT genotype were 1.00 and same as T allele frequency. The transition of C (cytosine) into T (thymine)on 1548 bp caused the lost of restriction site.


INTRODUCTION
Aceh cattle is one of Indonesian beef cattle which is suitable to be bred in Indonesia. This type of cattle adapts well to Indonesian environment, tropical climate, and able to live feedstuff (Sari et al., 2010). Even though Aceh cattle is able to adapt well, their productivity is still lower than other Indonesian local cattle. By improving the productivity of Indonesian local cattle, it is hoped that the breeders interest to breed local cattle will increase, so that the population of local cattle will increase and able to reduce Indonesian dependency on beef and cattle from other countries. The Indrapuri's Breeding and Forage Centre (IBFC) of Aceh cattle is a breeding station for Aceh cattle that was established since 2002. All Aceh cattle in this station is selected especially for producing elite bull, cow and heifer.
The GH gene is a single peptide of molecular weight equal to 22-kD secreted from pituitary gland in circadian and pulsatile manner, the pattern of which plays important role in postnatal longitudinal growth and development, tissue growth, lactation, reproduction, as well as protein, lipid and carbohydrate metabolism (Dybus et al., 2002). GH gene with its functional and positional potential has been widely used for marker in several livestock species including the Indonesian local cattle (Jakaria et al., 2009;Sutarno, 2010). Molecular genetic markers in animal breeding programs could make selection precise and efficient. Some of these markers are called candidate gene, e.g. the growth hormone genes, which are usually selected because of there biological significance on their quantitative traits of interest.
The study of GH gene MspI locus of Indonesian beef cattle have been reported in Ongole crossbred cattle (Sutarno et al., 2005;Sutarno, 2010), Madura cattle (Purwoko et al., 2003), Pesisir cattle (Jakaria et al., 2007), Bali cattle (Jakaria et al., 2009) and Grati dairy cows (Maylinda, 2011). Their studies indicated that polymorphism was found on Ongole crossbred cattle, Madura cattle, Pesisir cattle and Grati dairy cows. Bali cattle were monomorphic and the frequency of TT genotype and T allele were 1,00 as same as Ongole cattle in India (Lagziel et al., 2000). The research of GH gene MspI have never been reported in Aceh cattle. Based on description above it is necessary to carry out a research to identify the genotype of GH gene MspI in selected Aceh cattle at IBFC of Aceh cattle.

Blood Sample
The total number of blood samples were taken from 40 cattle at the breeding station of Aceh cattle. The blood sample was taken using venoject (EDTA) 5 ml on jugular vein, and then was kept in refrigerator (4 o C) for later laboratory analysis. The DNA isolation, extraction, amplification and digestion were all carried out in Laboratory of Animal Breeding, Faculty of Animal Sciences, Gadjah Mada University, Yogyakarta.

Genome DNA Extraction
Genome DNA extraction was carried out using Sambrook et al. (1989) method which was modified using buffer lysis cell on each sample containing of 15 µl 10% SDS, 6 µl aquabidest and 7.5 µl proteinase-K. The DNA was precipitated using 71 µl 5 M NaCl and 600 µl 96% ethanol. The precipitate was washed three times by adding 1ml 70% ethanol, then was centrifuged with the speed 12,000 rpm for 5 minutes. Then the DNA precipitate was dissolved in 100 µl ddH 2 O. The quality of the total genome was analyzed using 0.8% agarose gels electrophoresis (50 Volt, 15 minutes) in 1 x TBE and 10% ethidium bromide.

DNA Amplification
The DNA was amplified with Polymerase Chain Reaction (PCR). Each PCR reaction was made with the volume of 10 µl with the composition of 5 µl PCR master mix (KAPA, China); 0.5 µl primer forward and reverse; 0.5 µl DNA and 3.5 µl ddH 2 O. The forward primer was 5'-CCCACGGGCAAGAATGAGGC-3' and reverse primer was 5'-GAGGAACTGCAGGG GCCCA-3' (Mitra et al., 1995). The position of primer forward and reverse in PCR product (327 bp) of GH gene are shown in Figure 1. The PCR protocols to amplify the fragment were done by the initial denaturation temperature step at 94 0 C for 5 minutes for 1 cycle, followed by 30 cycles of denaturation at 94 o C for 1 minute, annealing at 60 o C for 50 seconds, elongation or extension at 72 o C at 1 minute and a final extension at 72 o C at 5 minutes (Zhou et al., 2005). The PCR product were separated by horizontal electrophoresis (50 Volt, 15 minutes) in 1% agarose gels in 1 x TBE and 10 % ethidium bromide.

Genotyping for GH Gene
The GH gene were analyzed by using the Polymerase Chain Reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) method. The PCR product of GH gene was Figure 1. The Position of Forward and Reverse Primer in PCR Product of GH Gene digested 37 o C for 3 hours by MspI restriction enzyme. Reaction consisted of 1.2 µl Buffer V2 10x; 8.7 µl ddH 2 O; 0.1 µl MspI restriction enzyme and 2 µl PCR product. Following the end of RFLP process, the products were then subsequently electrophorated (50 Volts, 30 minutes) using 1.5% agarose gel to identify polymorphism of allele. The MspI restriction enzyme recognized only the restriction site of four nucleotides for C↓CGG (Figure 2). The CC genotype consisted of two bands (105 bp and 223 bp), CT genotype consisted of three bands (105 bp, 223 bp, 328 bp), and TT genotype consisted of one band (328 bp).

Sequences of GH Gene Fragment
The PCR product of five cattle was used to sequence the GGH gene fragment. Each reaction consisted of 30 µl PCR product; 10 µl forward and 10 µl reverse primer (10 pmol/UI) repaired for sequencing process by Macrogen-BioSM Indonesia. Sequences of GH MspI gene was used to find nucleotide mutation in that fragment. The sequences of GH MspI gene was carry out in individual homozygote CC and TT.

Data Analysis
PCR-RFLP data was analyzed by allele frequency (Falconer and Mackay, 1996). The allele frequency was calculated by counting method as: Where p is the C allele frequency, q is the T allele frequency and N is the total number of cattle tested. The sequences was paralleled with the GH gene sequences from GenBank accessed code M57764 by alignment software (BioEdit and ClustalW).

Allele Frequency of GH MspI Gene
The DNA restriction fragments result was one genotype. Genotype of GH MspI gene showed 328 bp for TT genotype in all samples (Figure 3). Study of GH MspI gene in several worlds cattle reported that the frequency of T allele was higher in Bos indicus (hump) cattle group and lower in the Bos taurus (humpless) cattle group (Lagziel et al., 2000). The same research results also obtained that the frequency of T allele 1,00 in Bali cattle (Jakaria et al., 2009) and Ongole cattle (Lagziel et al., 2000). The T allele and TT genotype frequency of this research and other breed cattle in Indonesia are presented in Table 1.

Sequencing for GH MspI Gene
The samples managed to be sequenced were five samples from IBFC of Aceh cattle. The failure of sequencing was caused by unsuccessful amplification, limited number of DNA, and too many peak duplication on sequence graphic. The analysis on the diversity of nucleotide sequence was conducted using alignment program (BioEdit and ClustalW) after the sequence of Aceh cattle DNA was compared to the sequence of GenBank (accessed code M57764).
The change from C to T nucleotide in GH MspI gene nucleotide sequence in Aceh cattle was found on position 1548 bp and 1695 bp. The transition of C into T on position 1548 changed the MspI restriction site. This result is similar to that of Musa et al. (2013) Golpaygani and Sarabi (Zakizadeh et al., 2006). The length successful to sequence was from 1489 bp to 1772 bp (285 bp) on the third intron region. Based on this sequence results, it was concluded that several mutation found in this region. The variation of Single Nucleotide Polymorphism (SNP) which was identified in the third intron region from GH gene on several cattle are shown in Table 2. Sari et al. (2011) reported one new mutation on fifth exon on 2230 bp, in which C nucleotide turned into T nucleotide, and this was called silent mutation (CTC/Leu > CTT/Leu). Based on the sequencing result, it was concluded that the sequence of Aceh cattle on third intron was similar to Bos indicus group (Vechur, Butana, Kenana).