Polymorphism of Myostatin (MSTN) Promoter Gene and Its Association with Growth and Muscling Traits in Bali Cattle

Myostatin (MSTN) gene plays a key role in skletal muscle homeostasis such as inducing muscle athrophy, poliferation of myoblast, increasing ubiquitin-proteasomal, downregulating IGF pathway, and glucolysis. Myostatin gene expression is controled by CpG island located in promoter region. The objectives of this research were to identify polymorphism of MSTN promoter gene and to associate the polymorphism of SNP with growth and muscling traits in Bali cattle. A total of 48 Bali cattle from BPTU-HMT Bali island was screened to identify genetic polymorphisms in MSTN promoter region using sequencing method. The growth and muscling traits were measured at 12 months of age. The muscling traits were evaluated using ultrasound console with linear transducer having frequency 6.5 Hz and scaning we conducted at 130 mm in deep. Analysis of polymorphism was conducted by using PopGen 1.32 software. The association of MSTN with growth and muscling traits were analyzed by using General Linear Model (GLM) procedure. This result showed that a total of 20 polymorphic SNPs (seven SNPs in CpG island) were detected in this region. Although, only 3 SNPs (g.-8078C>T, g.-7996G>C, and g.-7930A>G) had equilibrium condition in Hardy-Weinberg analysis. The association result showed that 2 SNPs (g.-7799T>C and g.-7941C>T) were significantly associated with intramuscular fat percentage (P≤0.05) in Bali cattle. Although the 2 SNPs were nominally significant at nominal P≤0.05 threshold, they were not significant after Bonferroni correction for multiple testing. It could be concluded that MSTN promoter gene was polymorphic in Bali cattle and there were 2 SNPs associated with carcass quality.


INTRODUCTION
The myostatin (MSTN) gene is well-known as growth and differentiation factor 8 (GDF8) that is belonging to a member of transforming growth factor β (TGF-β super family). This gene consists of 3 exons and 2 introns (Kambadur et al., 2004). Myostatin gene has been located close to the centromere of bovine chromosome 2 (BTA 2) and encoded 375 amino acids then produce myostatin protein with 26 kDa molecular weight (Kambaduret al., 2004). Myostatin plays a key role in skletal muscle homeostasis such as inducing muscle athrophy, poliferation of myoblast, increasing ubiquitin-proteasomal, down regulating IGF pathway, and glukolisis (Elliott et al., 2012). The function of MSTN gene was an inhibitor (negative regulator) of proliferation and differentiation of cell cycle during myogenesis in embryonic and adult cell (Miyake et al., 2010). Kambadur et al. (2004) have identified that absence of myostatin affected increasing of skeletal muscle mass. This increment is due to a combination of hyperplasia (increasing muscle number) and hypertrophy (increasing muscle size).
Based on gene structure, MSTN has a CpG island, a region with repetitive of GC sequences that 70%-80% find in promoter of gene (Illingworth et al., 2010). The distribution of transcription initiation is usually over a region of 50-100 bp and there are appearance of the CpG island and lack of TATA box (Carninci et al., 2006).
The function of genome platforms for regulating transcription associated with promoter especially in a CpG island which has a role as genomic platforms to regulate transcription (Deaton et al., 2011). In addition, the CpG island was common for methylation among the region of the promoter attributing of chromatin condense and gene silencing (Sellner et al., 2007). Methylation of imprinted gene can increase or decrease the level of transcription, depending on a positive (suppressor) or negative (repressor) regulatory (Smith & Meissner, 2013). In vertebrates, the CpG island is distinct owing to their lack of DNA methylation and absences of CpG deficiency (Deaton et al., 2011). Mutation in CpG island of MSTN gene could change the regulation of expression via the generated CpG island and/or changed target sites for transcriptional regulator (Doherty et al., 2014). Mutation in MSTN gene in cattle showed different characteristics such as increasing of birth weight, higher muscling, faster growing, hyperplasia and hypertrophy in muscle (Kambadur et al., 2004). Previous research has identified MSTN gene in cattle intensively such as in Hanwoo cattle (Han et al., 2012), Qinchuan cattle (Zhang et al., 2007), Nellore cattle (Grisolia et al., 2009), Angus cattle (Gill et al., 2009), and Marchigiana cattle (Sarti et al., 2014).
Bali cattle (Bos javanicus) is one of Indonesian origin genetic resources that domesticated from bull (Bibos banteng) (Martojo et al., 2012). Bali cattle have potential to be beef cattle because well adapted in harsh environment, able to grow in marginal feed condition, high fertility and conception rate than other breeds (Purwantara et al., 2012). However, the utilization of Bali cattle is not optimal yet to produce meat in high quantity and quality. Improving Bali cattle quality by selection was conducted based on phenotypics data (conventional method) which has susceptibility that their environment impact. Selection using marker assisted selection (MAS) could be one of promising method for selection in cattle because it is more accurate, effective and effisien (Goddard & Hayes, 2007;Gorjanc et al., 2015). Therefore, improving Bali cattle genetic quality based on MAS using potential gene such as MSTN gene needs to be done on Bali cattle. The objective of this research was to identify the Single Nucleotide Polymorphism (SNP) of the MSTN gene in Bali cattle related to growth and muscling traits using direct sequencing method. The analyses of genotype and allele frequency were performed to elucidate polymorphism of this gene in Bali cattle. Association of MSTN with growth and muscling traits were also performed to identify significant SNP and its candidate for the genetic marker.

Animal and Phenotypic Data Source
A total 48 of Bali cattle were used (12-15 month of ages) in this study that consisted of 24 heifers and 24 steers from BPTU-HMT Denpasar, Bali Province. All of samples were risen in same paddock and feeding management. Each cattle was fed with grass (Pennisetum purpureum and Phaspalum notatum) in the amount of 10% of body weight and feed concentrate as much as 1% of body weight. The phenotypic variables that observed were growth traits including birth weight (BW), weaning weight (WW), yearling weight (YW), average daily gain (ADG), chest circumference (CC), body length (BL), and shoulder height (SH). Growth traits were measured based on BSN (2015). The muscling traits were evaluated using ultrasound console with linier transducer having frequency 6.5 Hz and scning were conducted with deep of 130 mm at transversal and longitudinal views. The muscling traits of ultrasound longissimus dorsi thickness (LDT), ultrasound back fat thickness (BFT), ultrasound rump thickness (RT), ultrasound rump fat thickness (RFT), ultrasound marbling score (MS) and Intramuscular fat percentage (PIMF) were assessed in this study. The measurement of LDT and BFT were carried out on the 12 th -13 rd ribs, two third from medial to lateral side models (Gupta et al., 2013;Melendez & Marchello, 2014) (Figure 1). The variables RT and RFT were measured between ileum and ischium (Silva et al., 2012) modified. In brief, the measurement of MS carried out according to AUS MET and MSA marbling reference standard. The percentage of IMF was carried out according to Deaton et al. (2000) on 12 th -13 rd ribs with region of interest by 30 x 30 mm. The image results were analyzed by using Image-J NIH software (ImageJ ®, NIH, USA) ( Figure 1). The general description of growth and muscling traits are shown in Table 1.

Genome Extraction and Amplification
Approximately 10 mL blood per cattle was collected aseptically from the jugular vein and kept in a tube containing anticoagulant of EDTA under temperature of 4°C. Genome extracted by using genomic DNA mini Kit (GeneAid DNA Ltd, Taiwan). The quality of total genome extractionswas performed by 1% agarose gel electrophoresis and was checked using spectrophotometry. The pairs of primer were used to amplify part of MSTN promoter gene. The forward primer: 5'-CCAACTATCCACCAGTAA-3' and the reverse primer: 5'-ACGACCAACCCTAACC-'3 were designed according to bovine MSTN gene (GenBank: AF348479.1) by using primer designing tool program (http://www. ncbi.nlm.nih.gov/tools/primer-blast/) and primer stat program (http://www.bioinformatics.org/sms2/ pcr_primer_stats.html). PCR reaction of MSTN gene was 50 µL consisted of 2 µL DNA sample, 22.6 µL distilled water, 0.2 µL forward and 0.2 µL reverse primers, and 25 µL GoTaq Promega Green MM. The PCR conducted in GeneAmp® PCR System 9700 Applied Biosystem Thermalcycler. Amplification condition consisted of predenaturation at 95°C 5 min followed by 35 cycles of denaturation at 95°C for 10 s, amplification at 63°C for 20 s, extention at 72°C for 30 s, and a final extension at 72°C for 5 min. The DNA amplification products were checked on 1.5% agarose gels in 0.5 x TBE running buffer and stained with EtBr then were visualized in UV trans-illuminator.

SNP Identification
Sequencing was performed for all of Bali cattle samples to define SNP in MSTN promoter region. Forward and reverse primer fragments were sequenced using sequencer machine (ABI Prims 3100-Avant Genetic Analyzer) in 1 st Base Selangor, Malaysia. The sequencing results were aligned using MEGA software (Tamura et al., 2011) to establish SNP. The BLAST (Basic Local Alignment Search Tool) program was used to search reference and homologous sequences in GenBank database.

Data Analysis
The genotypic and allelic frequencies from SNP, heterozygosity and Hardy-Weinberg equilibrium were calculated using PopGen program (Yeh et al., 1999). The association of MSTN gene and growth trait was analyzed by ANOVA PROC GLM and Duncan multiple ranget test (DMRT) procedure of SAS (SAS Inst., 2008). Furthermore, we also conducted Bonferroni correction for multiple testing. The statistical model used as follows the formulas below: where Y ijk is the mean value of the trait; µ is the general mean; α i is the fixed effect of MSTN genotype (i= 1, 2, 3); β j is the fixed effect of sex (j= 1, 2); ε ij is the random error.

Polymorphism of MSTN Promoter Gene in Bali Cattle
Result of PCR amplification consisting of 535 bp PCR products with 100 bpmarker was showed in    Table 2. Mostly, SNPs had 3 genotypes except SNP g.-8109T>G andg.-7905T>C, there were only homozygote genotype. The highest heterozygosity was found in SNP g.-7996G>C. In addition, the smallest heterozigosity were g.-8109T>G and g.-7905T>C having 0.000 value due to no heterozygote genotype found (Table 2). This re-search found that only 3 SNP in equilibrium condition, they were g.-8078C>T, g.-7996G>C, and g.-7930A>G (Table 2).

CpG Island Prediction in MSTN Promoter Gene
The sequence target of amplification was in promoter region which had CpG island (prediction using http://www.urogene.org/methprimer/). This prediction using criteria with minimum sequence length was >100bp, GC percentageis >50% and Obs/Exp ratio was >0.6. Seven mutation was found in CpG island region, they were g.
. This mutation might be affected the absences of CpG island in Bali cattle sequence (Figure 4).

Association Analysis
The association analysis showed that no growth traits were significantly associated with SNPs in MSTN promoter gene in Bali cattle (P<0.05) ( Table 3). In this result also showed that SNPs had no association with muscling traits, except 2 SNPs (g.-7799T>C and g.-7941C>T) which had significant effect on PIMF (P<0.05) ( Table 4). In the SNP g.-7799T>C, TT genotype has higher PIMF than CT and CC genotype in the SNP g.-7941C>T has higher PIMF than TT. Although the 2 SNPs

Polymorphism of Myostatin Promoter Gene
The SNPs in this research was polymorphic which represented by allele frequency lower than 0.99 or allel frequency higher than 0.01 (Nei & Kumar, 2000).
Based on chi-square (χ 2 ) analyses, only 3 SNPs in Bali cattle were in equilibrium condition (SNPs g.-8078C>T, g.-7996G>C, and g.-7930A>G). The factors which influenced Hardy-Weinberg equilibrium are non-random mating, selection, mutation, migration and genetic drift (Allendrof et al., 2013). Disequilibrium in Bali cattle genetic diversity might be caused by intensive selection, non-random mating, and mutation. Selection of Bali cattle in BPTU-HMT Bali province aimed to produce  superior bull. The mutation in Bali cattle is likely due to the efforts of these cattle to adapt harsh environmental conditions. The Ho value lower than He indicated that an inbreeding probability were occured in this population (Nassiry et al., 2009). Polymorphism in promoter region was also identified by He et al. (2013) at locus -371 of MSTN promoter gene which has 18 polymorphic SNPs in Qinchuan cattle. The most valuable meat is from longissimus dorsi which is in this research overall average of LDT and BFT were 33.047±5.077 mm and 1.455±0.348 mm, respectively. Putri et al. (2015) showed that LTD and BFT in adult Bali cattle (more than 3 years) was 57.577 mm to 63.818 mm and 1.935 mm to 2.324 mm, respectively.

Association of Single Nucleotide Polymorphism (SNPs) in Myostatin Promoter Region with Growth and Muscling Traits in Bali Cattle
The study revealed that there were no SNPs in promoter region of MSTN gene in Bali cattle had significant association with growth traits (P<0.05). Zhang et al. (2007) also found no significant association between MSTN promoter with birth weight, body weight at 6, 12, 18, and 24 mo. The lack of association between the SNPs in MSTN gene and growth traits was also found in Hanwoo cattle (Han et al., 2012). Furthermore, association of MSTN promoter gene showed no significant effect on morphological measurement as performed by Sarti et al. (2014).
Two SNPs in MSTN promoter region (g.-7799T>C and g.-7941C>T) showed significant association with PIMF (P<0.05) ( Table 4). Han et al. (2012) found association between MSTN promoter gene with meat quality index and fat colour index in Hanwoo cattle. The genotype of AA and AT in Hanwoo cattle had higher meat quality index and fat colour index than AA genotype. The expression of MSTN gene is inversely related to the other myogenic expression (Shibata et al., 2006). However, MSTN expression did not disturbed other myogenic expressions, such as Myog, Myf5, and MyoD and increased muscle mass. Futhermore, this mutation was able to reduce adiposity both of white fat and brown fat affected by neighboring muscle fiber (Li et al., 2015). Myostatin significantly inhibited differentiation of preadipocyte by cytokine from muscle fiber (Li et al., 2015). Moreover, they play cross role in muscle-fat which might regulate fat ratio in muscle such as IMF percentage (Sun et al., 2016). Promoter sequence was analysed in mammalian like cattle, pig, sheep, goat, human and mice. The mutation in TATA, CACCC, and AT1 has significantly decreased promoter activity, although mutation in AT2 and PAL likely to increase promoter activity (Allen & Du, 2008).

CONCLUSION
It could be concluded that myostatin (MSTN) promoter gene was polymorphic in Bali cattle and there were 2 SNPs (g.-7799T>C and g.-7941C>T) associated with carcass quality.