Identification of Myostatin Gene C.960delg Locus Polymorphism in Indonesian Local Sheep by Using Pcr-sscp Method

Myostatin (MSTN) is a protein inhibit muscle growth. This protein is a member of a superfamily ofmolecules called transforming growth factors beta (TGF-b). Deletion in c.960delG (1-bp deletion atposition 960) disrupts the reading frame from amino acid (aa) position 320 to ending in a premature stopcodon in aa position 359 have been found in Norwegian White Sheep. This deletion in the myostatingene is responsible to increase muscle mass, also known as 'double muscling', in sheep. The purpose ofthis study was to identify the polymorphism of myostatin gene in c.960delG locus of local sheep inIndonesia. The 832 DNA sampels from sheep were collected from 13 populations belonging to thePriangan (86), Javanese Thin Tail (389 i.e. Jonggol, Banjar, Ciomas), Javanese Fat Tail (94), West NusaTenggara (136), Rote Island/East Nusa Tenggara (35), Kisar Island/Southwest Maluku (22),Donggala/South East Celebes (45) and Batur cross breed/Wonosobo, Central Java (25). A gene fragmentof MSTN c.960delG length 299 bp was successfully amplified by using the technique of PCR(polymerase chain reaction) and genotyped by SSCP (single strand conformation polymorphism). Theresult showed no polymorphism in this gene. All sheep tested had G/G genotype for c.960delG locus.


INTRODUCTION
Local sheep has its own advantages to be protected and developed because it has several advantages, such as being able to adapt well in tropical environments, capable of lambing all year round, resistant to some kinds of diseases and parasites. Some weaknesses of the local sheep such as body weight and other body measurements have a very high diversity and the quality of meat has not yet met international market standards. Genetic improvement of local sheep through selection towards high productivity and quality of mutton is needed to be developed nationally due to the contribution of lamb on the national meat production is only 54,175 tonnes (2.5%) of total domestic meat production (Directorate General of Livestock Services, 2009). Progress in the field of molecular biology allows the selection efforts which can be made at the DNA level, that is by looking for genes that control the diversity of economic nature. Simple DNA test has to be applied to the detection of positive alleles at locus-locus economic value (ETL / Economic Trait Loci). Selection using a marker gene is an alternative livestock carrier biotechnology to produce desirable traits (according to the marker gene). Mapping genes in the sheep genome has marked the commencement of new horizons in the field of livestock breeding. The genes that can be used as genetic marker for selecting the properties of production and have high economic value are known as auxiliary selection markers or Marker Assisted Selection (MAS). Identification of genetic markers is an early and critical step to obtain markers auxiliary selection (MAS). Auxiliary selection markers have been shown to increase the genetic value of breeding livestock in the program as stated Parmentier et al. (2001).
At Texel sheep, Quantitative Traits Loci (QTL) markers for callipygian trait (increased muscle mass) have been identified in specific regions in chromosome number two (OAR2) (Marcq et al., 2002;Walling et al., 2004;Johnson et al., 2005). Mutation which occurs in the gene encoding myostatin (MSTN) has been found affecting the phenotype of increased muscle mass in mice (McPherron et al., 1997), cattle (Grobet et al., 1997), humans (Schuelke et al., 2004) and dogs (Mosher et al., 2007). Myostatin gene  in cattle has been mapped at the distal end of chromosome number two position by position 2q14-q15. The gene consists of three exons and two introns (Grobet et al., 1998). Research on myostatin gene has been reported by Boman et al. (2009) by using Norwegian white sheep and found a new point mutation in the 3 rd exon region. Mutation that occurs is 1-bp deletion at position 960 (position based on translational starting point) namely c.960delG. The deletion (c.960delG) disrupts the reading frame from amino acid (aa) position 320, ending in a premature stop codon in aa position 359. The incidence of deletion at this locus has not been reported in sheep in Indonesia. Therefore, the purpose of this study was to identify the deletion in c.960delG myostatin gene locus in Indonesian local sheep using PCR-SSCP method.

Blood Sample
Blood sample as a source of DNA were taken from vena jugularis by venoject needle with vaccutainer tube. The blood samples were used as much as 832 samples originating from 13 subpopulations belonging to the Priangan (i.e. fighting type 68 and meat type 18 samples), Javanese Thin Tail

DNA Extraction
DNA was extracted from blood in ethanol. Extraction procedure followed the phenolchloroform method (Sambrook and Russell, 2001) was modified by Andreas et al. (2010), with the following procedure: Sample preparation. Blood sample in the alcohol as much as 200 µl was poured to a 1.5 ml tube. Alcohol is 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.
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 measured.volume DNA molecules were deposited by adding a 2x volume of alcohol absolute and 0.1 x volume of 5M NaCl. The mixture was incubated at a temperature of -20°C during the night. Then, subsequent DNA precipitation was centrifuged at 12 000 rpm for 10 minutes. Obtained DNA precipitate was washed by 70% alcohol, and then precipitated again. Precipitated DNA clean from alcohol was restored by adding 100 µl TE (Tris EDTA). DNA samples were stored at -20°C and then was ready for use.

Amplification of MSTN c.del960G Locus
Primers to amplify MSTN c. 960delG locus followed Boman et al. (2009), with forward primer 5'-CTC CTT GCG GTA GGA GAG TG-3 'and reverse primer 5'-GGT GCA CAA GAT GGG TAT GAG-3'. The amplified product (amplicon) length was 299 bp. Amplification of MSTN c. 960delG locus was done by using PCR (polymerase chain reaction) methods. Reagents used for amplification of both of the target fragment are a 2 µl sample of 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™) was done with the condition of pra-denaturation at 94 ºC for 5 minutes, 35 cycles consisting of denaturation at 94 ºC for 45 seconds, annealing primers at 60 ºC for 45 seconds and extension of new DNA at 72 ºC for 1 minute, and the final extension at 72 ºC for 5 minutes.

Identification of Polymorphism MSTN gene c.del960G Locus by using PCR-SSCP Methods.
Identification polymorphism by using PCR-SSCP approach was performed to identify the c.del960G locus and another polymorphism in MSTN gene in Indonesian local sheep. PCR-SSCP method was used as an alternative method from previous study which used PCR-RFLP with MaeIII endonuclease (Boman et al., 2009). PCR-SSCP analysis was done by resolved the single pieces of DNA in PAGE (polyacrilamide gel electrophoresis) 12%. Electrophoresis process was conducted at 100 V for 17 hours. Visualized of single strand DNA bands were done by sensitive silver staining methods (Byun et al., 2009).

Sequencing and Sequence Analysis
Prior to sequencing, each band was cut out from PAGE and purified (Hu et al., 2010). This was then used as the DNA template for reamplification then sent to sequencing platform (Macrogen Inc., Korea). Ensuring that amplicon product are specific, BLAST (basic local alignment search tool) methods was used to calculate sequences homology with GenBank database (http://www.ncbi.nlm.nih.gov/BLAST). Alignments and comparisons of sequence were carried out by using MEGA software version 4.0.

Genotyping 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. Mathematics model genotype and allele frequency (Nei and Kumar, 2000) are represented as follows: Note : χ ii = ii th genotype frequency n ii = number sample of ii genotype n ij = number sample of ij genotype N = total sample χ i = i th allele frequency

Amplification of MSTN c.del960G region
Amplification of MSTN c.del960G locus was carried out on GeneAmp® PCR System 9700 (Applied Biosystems™) with annealing in the same temperature, which was 60°C. The amplified gene fragments were visualized on 1.5% agarose gel (Figure 1). The amplified product (amplicon) length MSTN c.del960G fragment was 299 bp, including a part of 2 nd intron and a part of 3 rd exon (Boman et al., 2009).

Identification of MSTN c.del960G by using PCR-SSCP Methods
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 change 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).
Given its relative technical simplicity and high mutation detection capacity, the PCRcoupled SSCP provides a powerful tool to specifically identify pathogens and to screen effectively for genetic variability within and among samples, specimens or populations, and importantly, to screen for unknown mutations (Sunnuck et al., 2000;Doi et al., 2004;Jespersgaard et al., 2006;Tahira et al., 2006). This approach is relatively simple technically and can be used effectively to visually display at different sequence types within and between amplicons (of ~ 100-500 bp) and to rapidly screen hundreds of samples per day (Gasser et al., 2007).
It remains possible that, occasionally, some point of mutations are not detected because of the nucleotide composition (e.g. A+T or G+C richness) or excessive length of a DNA region being analyzed (Gasser et al., 2007). The mutation detection rate of SSCP varies depending on the parameters, such as size of the fragment, base composition of the sequence, electrophoresis temperature and/or gel composition (i.e. pore size and cross-linking) (Hayashi, 1991;Sunnuck et al., 2000;Doi et al., 2004;Jespersgaard et al., 2006;Tahira et al., 2006;Gasser et al., 2007).
Migration patterns of DNA single strand band in PAGE 12% produced four MSTN c.960delG DNA single strand banding patterns ( Figure 2). The four patterns of these bands were uniform for all samples in this study. For the purposes of sequencing analysis, each band was cut from PAGE and then purified using TE wash buffer method (Hu et al., 2010). The results of purifications was used as a sample for reamplification process and sent to sequencing platform (Macrogen Inc., Korea).

Sequence Analysis
Sequence analysis revealed that amplicons were 299 bp in length. These were teh same size as previous study reported by Boman et al. (2009). There was not polymorphism identified from different banding pattern. All of four banding pattern in PCR-SSCP represented the same allele. Genotype found in buffalo in this research was G/G genotype. Occasionally, one strand is separated into two or more bands in the SSCP gel, eventhough the sequence is the same. This suggests that the strands having the same sequence can have different stable conformations. Usually, intensity of these conformer bands may be different, but the ratio of the intensity between them is constant from sample to sample. Therefore, such sequence-different conformation situations are easily diagnosed (Hayashi, 1991).
All of our sequences in this study have very high similarity (100%) with published in ovine myostatin gene sequences (GenBank Accession no. DQ990914). There did not reveal either of any deletion in 1-bp deletion at position 960 (position based on translational starting point, Boman et al., 2009) or another mutation from this region in all populations Indonesia Local Sheep (Figure 3).

Genetic Diversity of MSTN c.del960G Locus within Indonesian Local Sheep
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 MSTN c.del960G locus are presented in Table 1.
Genetic diversity based on molecular marker MSTN c.del960G locus in Indonesia local sheep are very low. This is indicated by the value of one genotype frequency and allele which has a value of 1, which marks the fixation process. This value are lower then reported by Sumantri et al. (2008) and Dagong et al. (2011)   adaptation process which suggested that the animal which can survive in this enviromental are having small performance. In this case, the presence of the c.960delG mutation in both alleles means that no functional myostatin protein is expected to be produced (Boman et al., 2009). If myostatin not to be expressed, so the negative growth regulation will failure and will increase the number of muscle fibers (hyperplasia).

CONCLUSION
Based on these results, it can be concluded that the diversity of MSTN c.del960G locus in Indonesian local sheep is very low and showed no polymorphisms were detected in this gene. All of sheep were tested had GG genotype for MSTN c.del960G locus. So, MSTN c.del960G locus can not be used as marker to assist selection process in Indonesian local sheep.

ACKNOWLEDGMENTS
The work was supported by Directorate General of Higher Education, Ministry of Education Republic of Indonesia through the    (68)