Association of SNP g.643G>A of MYF5 gene polymorphism with body weight and body measurements in Bali cattle

This study aimed to identify the SNP g.643G>A of MYF5 gene then associate it with body weight and body size measurements in Bali cattle. Blood samples were collected from 80 bali cattle at BPTUHPT Denpasar Bali. Data on phenotypic properties observed included; birth weight, live weight, average daily gain, body length, chest depth, withers height, hip height, and heart girth. Polymorphism of the MYF5 gene was identified using the PCR-RFLP method. Association of MYF5 genotypes with body weight and body size measurements was performed using General Linear Model by SAS 9.4 program. MYF5│MspI gene was polymorphic with three genotypes: AA, AG, and GG. Genotype frequency of AA, AG and GG were 0.04 (3), 0.30 (24), and 0.65 (53), respectively. Allele frequencies were 0.19 and 0.81 for A and G alleles, respectively. Gene frequency analysis showed that Bali cattle at BPTU-HPT Denpasar was in Hardy-Weinberg Equilibrium. Association of MYF5│MspI gene with body weight and body measurement were not significantly different. SNP g.643G>A could not be used as a genetic marker for the body weight and body size measurements in Bali cattle.


INTRODUCTION
Indonesia is a maritime country consists of many Islands and is rich in livestock genetic resources, one of them is Bali cattle (Mohamad et al., 2009). Bali cattle (Bos javanicus) is one of the native Indonesian cattle produced by domestication of the bull (Bibos banteng) and has been recognized by Food and Agriculture Organization (FAO) as one of the world's cattle nations that lives in the Indonesian region (Martojo, 2012). As a national animal genetic resources, Bali cattle should be maintained and utilized productively (Sutarno and Setyawan, 2016).
Bali cattle is adapted to the Indonesian climatic conditions and is able to be maintained intensively or extensively and still gives high production (Sutarno and Setyawan, 2016). This cattle has high conception rate (70-90%), high carcass percentage (45-57%) (Purwantara et al., 2012). National meat requirements are mostly met by local Indonesian beef production. Indonesia's local cattle population in 2017 was estimated to be 16 599 247 heads and continues to grow by 3.59%. One of the local cattle that contributes to the fulfillment of national meat needs is Bali cattle. Even though it has superior potential, the use of Bali cattle genetic resources is not optimal, especially in terms of genetic quality. One approach to improve genetic quality and productivity is through a selection method (Supriyantono et al., 2012).
Rapid development of DNA-based molecular technology has become one of the selection methods that uses genetic markers known as Markers Assisted Selection (MAS). Selection in bali cattle can be done on high economic traits. One of them is the of growth rate (Supriyantono et al., 2012). The growth rate is controlled by many genes (Casas and Kehrli, 2016). One potential gene that plays a role in growth is the MYF5 gene (Chung and Kim, 2005;Nasr et al., 2016).
The MYF5 gene in cattle is mapped on chromosome 5 which consists of 3 exons and 2 introns (Seong et al., 2011). The MYF5 gene is a member of Myogenic Regulatory Factors (MRFs) which is expressed the earliest among members of other MRFs and one of the genes involved in the mechanism of skeletal muscle formation and the formation of lateral sclerotome derivatives (Hernández-Hernández et al., 2017). MYF5 gene has an important role in myocyte determination and differentiation (Li et al., 2004) and also controls myogenesis and myoblast growth during prenatal embryo development (Guo et al., 2015). Myogenesis also controls the expression of some myogenic regulatory factors (MYOD and MYF5) (de las Heras-Saldana et al., 2019).
Several studies related to the SNPs identification of the MYF5 gene have been carried out widely in several livestock such as hanwoo cattle (Chung and Kim, 2005), Canada cattle (Li et al., 2004) Jiaxian cattle, Qinchuan cattle, Luxi cattle, Nanyang cattle and Xianan cattle (Ujan et al., 2011). This gene was found to be significantly associated with yearly body weight (Li et al., 2004) and body gain (Chung and Kim, 2005). Some researches were conducted on MFY5 gene in bali cattle but are still few especially on MYF5 gene polymorphisms and its association with growth traits. Bali cattle is one of the livestock species used to provide meat in Indonesia. Therefore, identification of SNP g.643G>A of MYF5│MspI in bali cattle has important role in increasing meat production in Indonesia. The present study aimed to identify MYF5│MspI gene polymorphisms and its association with body weight traits and body size measurements in bali cattle.

Animals and Samples
This research was conducted at BPTU-HPT Denpasar of Bali province and at the Molecular Genetic Laboratory of Animal Science, Department of Animal Production and Technology, IPB University (Bogor Agricultural University), Indonesia. Blood samples for DNA extraction were collected from 44 female and 36 male Bali cattle at jugular vein using venoject needle.

DNA Extaction and Polymerase Chain Reaction-Rectriction Fragment Length Polymorphism (PCR-RFLP)
DNA extraction was performed using Geneaid DNA extraction kit procedure (Geneaid Biotech Ltd). PCR was performed for amplification of polymorphism region of MYF5 gene. A pair of primers used was designed by (Ujan et al., 2011). These primers (forward and reverse) were 5'-ACGACCAACCCTAACC-3' and 5'-CCAACTATCCACCAGTAACC-3', respectively. They were used to amplify a 285 base pairs fragment according to the beef cattle genomic sequence in the GenBank database (accession number M95684.1). The PCR was performed under the following conditions, initial denaturation at 95 ˚C for 5 minutes and for 1 cycle. The second phase consisted of 35 cycles, each cycle consisting of denaturation process at 95 ˚C for 10 seconds, primer annealing at 51 ˚C for 20 seconds and DNA extension at 72 ˚C for 30 seconds. The final phase was the primer elongation or final extension at 72 ˚C for 5 minutes. The DNA amplification product of 285 bp was visualized by 1.5% agarose gel electrophoresis.
PCR products from polymorphic region of MYF5 gene (285 bp) were digested with MspI restriction enzyme selected according to the software (http://tools.neb.com/NEB cutter2/index.php) of the polymorphic site. PCR product and MspI restriction enzyme were incubated at 37 ˚C for 16 hours (Thermo Fisher Scientific, EU, Lithuania). The product of DNA fragments from PCR-RFLP were visualized using agarose gel electrophoresis with a concentration of 2%, 0.6 agarose powder was added to 30 ml of 0.5 x TBE, the mixture then heated to boiling and added to 1 uL florosafe electrophoresis was run on average voltage of 100 volts for 35 minutes. Gels were visualized under ultra-violet transilluminator (Alpha Imager, Alpha Innotech, Santa Clara, USA).

Genotype Frequency, Allele Frequency and Hardy-Weinberg Equilibrium
Genotype and allele frequencies, chi-square and Hardy-Weinberg Equilibrium (HWE) were calculated using Popgene32 software program (Yeh et al., 2000).

Association of MYF5 Gene Polymorphisms with Body Weight and Body Size Measurements
The MYF5 gene polymorphisms and association with body weight traits and body size measurements was analyzed using General Linear Model (GLM) (SAS 9.4). The following statistical model was used: Y ijk = µ + α i + β j + γ k + Є ijk Where: Y ijk = number of observed; µ = mean; α i = effect of i genotype; β j = sexs effect; γ k = effect of age: cattle were grouped according to their age (1.5 -2 years; 2 -2.5 years and 2.5 -3.5 years); Є ijk = random errors. Least square means (LSM) was used to test the significance difference among genotypes.

MYF5 Gene Polymorphism
The MYF5 gene Single Nucleotide Polymorphism (G>A) is located at the nucleotide position g.643, exon 1 of chromosome 5 with GenBank Accesion Number: M95684.1. The 285 bp fragment of MYF5 (g.643G>A) gene was successfully amplified in Bali cattle using Applied Biosystems PCR machine under conditions of annealing temperature 51˚C for 20 seconds as shown in (Figure 1).
The MYF5 fragment of 285 bp was digested with MspI restriction enzyme and resulted into three genotypes designed as AA (a single, uncut fragment of 285 bp), GG (two fragments of 115  (Figure 2). MYF5│MspI for Bali cattle at BPTU-HPT Denpasar is polymorphic. Polymorphism occurs when each allele frequency is greater than 0.01 (Allendorf and Luikart, 2007). The restriction enzyme recognizes DNA fragment and cuts it at specified position, while visualization of PCR-RFLP results was carried out using 2% agarose gel.
Genotype frequencies of AA, AG and GG are, respectively 0.04 (3), 0.30 (24) and 0.65 (53) and allele frequencies are presented in Table 1. Allele G was more frequent with 0.81 and 0.19 for allele A. Gene frequency analysis showed that Bali cattle population at BPTU-HPT Denpasar was in Hardy-Weinberg equilibrium as shown in Table 1. Chen et al. (2017) stated that Hardy-Weinberg equilibrium occurs in a larger population size, if the frequency of dominant and recessive genotypes is constant from generation to generation, there is no selection, mutation, migration, and genetic drift. Selection is one of the factors that can quickly change the equilibrium in the population. The present study showed that there was no selection in Bali cattle reared at BPTU-HPT Denpasar.

Association of MYF5 Gene Polymorphism with Body Weight and Body Size Measurements of Bali Cattle
Association analysis of SNP g.643G>A MYF5│MspI gene with body weight and body size measurements showed that the SNP studied was not significantly associated with body weight and body size measurements (P>0.05). Results are presented in Table 2. Although mutation occurs in exon, SNP g.643G>A did not cause a significant effect because the mutation was synonymous. The SNP at g.643G>A position with CCG or CCA codon will translated to proline amino acid. This SNP is specific for Bali cattle, and it is not found in other Indonesian beef cattle. Chung and Kim (2005)   gene. This mutation was clearly associated with body weight of 12 months and daily body weight gain in Korean cattle. Seong et al. (2011) also reported that the MYF5 intron 2 gene in the SNP A1948G had a significant effect on the back fat thickness and a 6 month live weight in Hanwoo cattle. SNP of MYF5 gene that are associated with growth properties can be used as the genetic markers. In the future, the identification of MYF5 gene SNPs in cattle must be continued. It is expected that the use of genetic markers can be applied as an assisted selection marker to improve the genetic quality of Bali cattle.

CONCLUSION
SNP g.643G>A on the MYF5│MspI gene was polymorphic. The population of Bali cattle at BPTU-HPT Denpasar was in Hardy-Weinberg equilibrium. Association of the polymorphism of the MYF5│MspI gene in Bali cattle did not show a significant difference on body weight and body size measurements of Bali cattle.

ACKNOWLEDGEMENT
This work was financially supported by PDUPT Research Grant from Ministry of Research, Technology, and Higher Education, Republic of Indonesia, number: 1757/IT3.11/PN/2018. We also appreciate to Head of BPTU-HPT Bali cattle, Bali Province.