Identification of Gh|alui and Ghr|alui Genes Polymorphisms in Indonesian Buffalo

Growth hormone (GH) is an anabolic hormone which sintesized and secreted by somatrotop cell inpituitary anterior lobe. GH exert its effect on growth and metabolism by interacting with a specificreceptor on the surface of the target cells. Growth hormone receptor (GHR) has been suggested ascandidate gene for traits related to meat production in Bovidae. The objectives of this study were toidentify polymorphism of GH and GHR genes in buffalo. The 452 DNA samples buffalo were collectedfrom five populations in Indonesia (Siborong-Borong-Medan (65), Lebak-Banten (29), Pandeglang-Banten (180), Semarang-Central Java, and Mataram-West Nusa Tenggara (103)). A gene fragment of theGH|AluI gene at 432 bp located on exon 3 and GHR|AluI gene at 298 bp on exon 10 were successfullyamplified by using the techniques of a PCR (polymerase chain reaction) and genotyped by PCR-RFLP(restriction fragment length polymorphism) then -SSCP (single strand conformation polymorphism). Theresults showed no polymorphisms were detected in these genes. All buffaloes tested had LL genotype forlocus GH|AluI and AA genotype for locus GHR|AluI.


INTRODUCTION
Authorized national livestock sector development is largely determined by the ownership, preservation and utilization of biological resources such as livestock animals that have been developed and are still maintained subsistence. Buffalo (Bubalus bubalis) is one large ruminants that has its own advantages for development because it can survive with lowquality of feed, tolerant to local parasites and such existence has been fused with social and cultural life of Indonesian farmers. Buffaloes maintained by the farmer in the traditional way used as a draft, meat production, organic fertilizer, leather and socio-cultural ritual. Thus, the local buffalo is a source of germplasm that can be used in order to increase food availability, to improve public welfare, to create employment and to generate foreign exchange. Animals that are genetically adapted to specific environmental condition, would be more productive because it can be developed using low cost, supporting the diversity of food, agriculture and culture, as well as effective in achieving the objectives of food security (FAO, 2000).
Growth hormone (GH) is an anabolic hormone synthesized and secreted by cells of lobe somatotrop in anterior pituitary (Ayuk and Sheppard, 2006). GH has an important role in the growth and postnatal development, growth tissue, lactation, reproduction, and proteins, lipids and carbohydrates metabolism (Akers, 2006;ThidarMyint et al., 2008). GH gene diversity in Japanese Black cattle influenced carcass characteristics and fatty acid composition (Ardiyanti et al., 2009). Growth hormone receptor (GHR) is a transmembrane protein that binds GH by high affinity and specificity. Receptor expression is required to produce the cellular activity of GH. This indicates that the changes in GHR function can affect the ability of GH binding and GH activity in target tissues (Di Stasio et al., 2005).
Information of diversity using molecular approaches at the local buffalo in Indonesia is still very rare. Diversity of functional genes has been widely used as an auxiliary marker selection on some livestock commodities, combined with optimal maintenance management. The aim of this study was to gather information GH and GHR gene diversity at the local buffalo in Indonesia.

DNA Sample
DNA samples obtained from blood and buffalo meat. The blood samples were used as a source of as much as 320 DNA samples originating from five different regions, namely 65 samples from Siborong-Borong (North Sumatera Province), 29 samples from Lebak (Banten Province), 48 samples from Pandeglang (Banten Province), 75 samples from Semarang (Central Java Province), and 103 samples from Mataram (West Nusa Tenggara Province), while the samples that were used as a source of meat as much as 132 DNA samples were from Pandeglang (Banten Province).

DNA Extraction
DNA was extracted from blood and buffalo meat. Extraction procedure followed the phenolchloroform method (Sambrook and Russell, 2001) was modified, with the following procedure:

Sample preparation.
Meat samples within alcohol were as much as 70 mg, whereas the blood in the alcohol were as much as 200 µl. Sample was inserted to a 1.5 ml tube. Alcohol was eliminated from the sample by adding distilled water until 1000 µl, and left in room temperature for 20 minutes. Then it was precipitated by centrifugation at a speed of 8000 rpm for 5 minutes.

DNA precipitation
Samples were centrifuged at a speed of 5000 rpm for 10 minutes to separate the water phase with phenol phase. Water phase was transferred in a new tube with the volume measured. DNA molecules deposited by adding a 2x volume of alcohol absolute and 0.1 x volume of 5M NaCl. Then the mixture was incubated at a temperature of -20 °C over night. Subsequent DNA precipitation was by centrifugated at a speed of 12 000 rpm for 10 minutes. Obtained DNA precipitate was washed by 70% alcohol, and then precipitated again. Precipitated DNA clean from alcohol restored by adding 100 µl TE (Tris EDTA). DNA samples were stored at -20 °C and ready for use.

Amplification of GH and GHR Genes
Amplification of GH and GHR fragment were done by using PCR (polymerase chain reaction) methods. Reagents used for amplification of both target fragment were a 2 µL sample DNA, each primer 25 pmol, 200 µM dNTPs mixture, 1 mM MgCl2, and 0.5 units of DreamTaq™ DNA Polymerase and 1x buffer (Fermentas) in total solution 25 µL. Amplification in vitro within GeneAmp® PCR System 9700 (Applied Biosystems™) done with the condition of pra-denaturation at 94ºC for 5 minutes, 35 cycles consisting of denaturation at 94ºC for 45 seconds, annealing primers at 62ºC for 45 seconds and extention of new DNA at 72ºC for 1 minute, and the final extention at 72ºC for 5 minutes.

Genotyping by using RFLP Method
Determination of genotypes of each individual was done by using restriction fragment length polymorphism (RFLP), follow by visualized on 2% agarose gel with 0.5 x TBE buffer (tris borate EDTA) at 100 V for 40 minutes. Gel was stained by ethidium bromide, and visualized on UV transiluminator. Cutting enzyme that is used for both sides of the target gene was AluI.

Identification of Polymorphism by using PCR-SSCP and Sequencing Methods
Diversity detection with PCR-SSCP approach performed as an alternative in the identification of GH and GHR gene diversity related to quality of buffalo meat. PCR-SSCP analysis was done by resolved the single pieces of DNA in PAGE (Polyacrilamide gel electrophoresis) 8%. Electrophoresis process was conducted at 100 V for 16 hours. Visualized of single strand DNA bands were done by sensitive silver staining methods. In addition to PCR-SSCP, the eight samples were sequenced to indentify the difference DNA sequence of GH and GHR gene were observed by DNA sequence GenBank.

Genotype and Allele Frequency
Genotype frequency represents the ratio of a genotype to total population. Allele frequency is a ratio of an allele to the overall allele at a locus in the population. Mathematical model genotype and allele frequency (Nei and Kumar, 2000) is represented as follows: where : x ii = ii th genotype frequency n ii = number sample of ii genotype n ij = number sample of ij genotype N = total sample x i = i th allele frequency

Amplification of Buffalo GH and GHR Genes Fragment
Amplification of GH and GHR genes fragment was carried on GeneAmp® PCR System 9700 (Applied Biosystems™) with temperature of 62°C. The amplified gene fragments were visualized on 1.5% agarose gel (Figure 1). The amplified product (amplicon) length GH gene fragment was 432 bp, including 55 bp of 4 th exon, 4 th intron, and 99 bp of 5 th exon (Balogh et al., 2009). The amplicon length of GHR gene fragment was 298 bp located in 10 th exon (Genbank Access No. AY053546).

Identification of GH and GHR Genes by Using PCR-RFLP Method
Determination of GH and GHR gene genotypes in this study was done by PCR-RFLP method using AluI which have cutting site AG|CT. Based on DNA sequences of GH genes amplified segment there were three sites AluI cutting, which produced fragments of length 20, 51, 96, and 265 bp, known as the leucine allele (L). There was a substitution from C to G at position 1758 (Lucy et al., 1993), so the produces fragments of length 20, 147, and 265 bp, known as the valine allele (V) (Balogh et al., 2009). Visualization on 2% agarose gel showed that the GH|AluI locus the fifth buffalo population was monomorphic. The LL genotype were found in a total sample (Figure 2).
Restriction by using AluI enzyme done on the amplicon of GHR gene fragment (GHR|AluI) produces fragment of length 81 and 217 bp, known as the A allele. Some studies showed the substitution at position 256 in cattle (Genbank accession number AY053546), from A to G. These changes caused a loss of enzyme recognition sites of AluI, so that produced fragment of the length 298 bp, known as the G allele (Ge et al., 2000;Di Stasio et al., 2005). Genotype found in buffalo in this research was AA genotype (Figure 3).

Genetic Diversity of GH|AluI dan GHR|AluI Genes within Indonesian Buffalo
Genetic diversity within a population can be used as a parameter in studying the population and evolutionary genetics, in addition, it can be used to identify and preserve the peoples in the population associated with the character of a special nature. Knowledge of the genetic diversity of a nation will be very useful for food security and continuous availability (Blott et al., 2003).
Level of diversity within populations can be drawn from the allele frequency. Allele frequency is a ratio of one allele relative to the overall allele found in one population. Information on genetic diversity of a population using multiple loci, can be described by the value of heterozygosity (Nei and Kumar, 2000). Genotypes and allele frequencies of GH|AluI locus is presented in Table  1, while for the GHR|AluI locus is presented in Table 2.
Genetic diversity based on molecular marker GH|AluI and GHR|AluI loci in buffalo were very low. This was indicated by the value of one genotype frequency and allele which had a value of 1, which marks the fixation process. Diyono (2009)   limited number of males in the population, and the high inbreeding frequency. The number of samples of male buffalo found in this study (20% of the total sample) was less when compared to samples of female buffaloes. The number of albino buffalo, can be used as one indicator of the high frequency of inbreeding. Nei and Kumar (2000) argued that the high frequency of inbreeding can reduce the diversity in the population.

Identification of GH and GHR Genes by using PCR-SSCP Methods and Sequencing
Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) is one further analysis method that utilizes PCR product. PCR-SSCP method is a reliable method of quickly detecting a mutation (Hayashi, 1991). This method is based on the assumption that the nucleotide acid changes would lead to changes in migration patterns on polyacrylamide gel nondenaturasi (Barroso et al., 1999). Mutation was detected from the differences in migration patterns from conformation of single strand DNA on polyacrylamide gels (Hayashi, 1991). Migration pattern of single strand DNA of GH and GHR gene fragment on PAGE in this study were uniform.
Analysis of nucleotide similarity of GH and GHR gene sequencing results with the sequence of nucleotides in cattle and buffaloes in Genbank was done by BLAST method (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence of buffalo GH gene (Figure 4) did not reveal any base change from C to the G at position 1758 (Lucy et al., 1993;Balogh et al., 2009). The same thing happened to sequence of GHR gene fragment (Figugre 5), which did not reveal any base changes from A to G at position 256 (Ge et al., 2000;Di Stasio et al., 2005).

CONCLUSION
It can be concluded that the diversity of GH| AluI and GHR|AluI genes in Indonesian buffalo was very low and showed no polymorphisms were detected in these genes. All buffaloes tested had LL genotype for locus GH|AluI and AA genotype for locus GHR|AluI.