Polymorphism of the myostatin gene exon 1 using PCR-RFLP technique in five beef cattle in Indonesia

This study aims to analyze the polymorphism of the myostatin (MSTN) gene in exon 1 at SNPc.111G>C (rs523392653) and SNPc.267G>A (rs383271508) on various beef cattle in Indonesia. A total of 136 DNA samples were analyzed in this study, consisting of 33 heads of Madura, 31 heads of PO, 36 heads of Bali, 18 heads of Simmental, and 18 heads of Limousin cattle. The DNA sample was collected from the Animal Molecular Genetic Laboratory, Faculty of Animal Science, IPB University. Along 608 bp of DNA sequence was amplified using PCR with forward primer was 5′-CAA GTT GTC TCT CAG ACT GG-3′ and reverse primer was 5′-CTC CTC CTT ACA TAC AAG CC-3′ and annealing temperature was 59°C. The amplified DNA was then digested with HaeIII and AluI restriction enzymes using the RFLP method to determine polymorphism. Genotype frequencies, allele frequencies, PIC, and heterozygosity values were calculated with the PopGen32 program. The HaeIII enzyme produces two genotypes, GG and CG, while the AluI enzyme produces three: GG, AG, and AA. SNPc.111G>C polymorphism was found in Madura and PO cattle, while SNPc.267G>A polymorphism was found in Madura, PO, and Bali cattle. Both Simmental and Limousin cattle were monomorphic.


Introduction
Amino acids are molecules used by all living things to make proteins.Meat is one food source containing various types of amino acids essential for the body [1].In Indonesia, the domestic demand for meat is still primarily fulfilled by importing fresh meat or beef cattle from several countries, such as India, Brazil, and Australia.The total import of fresh meat from these three countries is estimated to be 223,000 tons [2].The importation of beef cattle is caused by domestic production, which can only fulfill 60% of the demand [3].Domestic meat demand can be fulfilled by utilizing the genetic resources of beef cattle available in Indonesia.Indonesia also has many breeds of beef cattle that have economic value and are well-adapted, allowing for further development.
Several beef cattle breeds that have long adapted in the Indonesian marginal region are found, such as Bali cattle [4], PO cattle, and Madura cattle [5].Some cattle from the Bos taurus species are also raised in Indonesia, such as Simmental and Limousin cattle [6].Nevertheless, beef cattle productivity in Indonesia remains lower than that of foreign cattle.One approach to improving and enhancing local livestock's genetic quality is through selecting programs [7].Currently, the breeding program in cattle is still widely practiced conventionally.Conventional selection leads to relatively slow genetic progress due to its reliance solely on phenotypic data and needs long generation intervals.Comprehending carcass and meat quality plays a vital role in the beef market and critically influences the trajectory of diverse beef production systems.Consequently, investigating the effects of contributing genes to seek the 2 differences in linkage to carcass and meat quality attributes in distinct beef cattle breeds is necessary [8].
Muscle growth is a complex characteristic influenced by multiple gene pairs and environmental factors [9].The Myostatin gene is located on chromosome 2 of cattle and consists of three exons and two intronic regions.Its codes region spans 1,128 nucleotides, resulting in a protein length of 375 amino acids [10].Among these genes, the myostatin gene regulates muscle development and growth traits [11].Previous studies have identified two mutations in the MSTN gene on exon 1, namely SNPc.111G>C (rs523392653) and SNP.267G>A (rs383271508) [10].One of the technologies that has been long used in the field of molecular genetics is PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism).PCR-RFLP is a technique that is widely used because it is faster, cheaper, more accessible, reliable, and highly accurate in determining a genotype [12].This study aims to analyze the polymorphism of the myostatin (MSTN) gene at SNPc.111G>C and SNPc.267G>A of several beef cattle breeds in Indonesia such as Madura, PO, Bali, Simmental, and Limousin cattle using PCR-RFLP technique.

DNA sample collection
A total of 136 DNA samples were analyzed in this study.The DNA samples used in this study were collections from the Laboratory of Genetics Molecular, Faculty of Animal Science, IPB University, consisting of 33 heads of Madura cattle from Madura Island, 31 heads of PO cattle from Bogor at Cipelang breeding station, 36 heads of Bali cattle from Denpasar and Nusa Tenggara Timur, 18 heads of Simmental and 18 heads of Limousin cattle from Padang Mangatas at BPTU-HPT breeding station.All of those DNA samples were isolated from fresh blood.

Genotyping of MSTN gene using PCR-RFLP method
The DNA samples were analyzed at the Laboratory of Genetics Molecular, Faculty of Animal Science, IPB University.The primer used to amplify the MSTN gene in exon 1 was followed by previous research [10].A fragment of MSTN exon 1 sequence of each cattle was amplified by forward primer was 5′-CAA GTT GTC TCT CAG ACT GG-3′ and reverse primer was 3′-CTC CTC CTT ACA TAC AAG CC-5′ (Figure 1).A total of 15.0 µL of PCR mix was obtained by mixing 1.0 µL of DNA isolation, 7.5 µL of Red Mix, 0.2 µL of reverse primer, 0.2 µL of forward primer, and 6.1 µL of nuclease-free water (Qiagen, Germany).The reaction was run in the AB systems Geneamp 9700 Thermal Cycler (Conquer Scientific, Inc., CA) under the following conditions: 1 minute of predenaturation at 95 o C (1 cycle); 15 seconds of denaturation at 95 o C (35 cycles); 20 seconds of annealing at 59 o C (35 cycles); 10 seconds of extension at 72 o C (35 cycles); and 5 minutes of final extension at 72 o C (1 cycle).The PCR result was observed through electrophoresis conducted in 1.5% of agarose gel (Vivantis Inc., USA) and stained by Flourosafe (Axil Scientific Pte Ltd., Singapore), then immersed in a 0.5X TBE (Tris Boric EDTA) solution.The length of the band was measured using a 100 bp DNA marker (Geneaid, Taiwan).A UV transilluminator (AlphaImager, USA) observed the electrophoresis result.
The MSTN gene was analyzed using the PCR-RFLP technique.The amplicon (608 bp) of the MSTN gene was digested at 37 o C for 4 hours with HaeIII (5'-GG│CC-3') to identify the SNPc.111G>C and AluI (5'-AG│CT-3') for SNPc.267G>A that determined by Neb-cutter program (https://nc3.neb.com/NEBcutter/).Both HaeIII and AluI enzymes had the same volume reaction mix performed by 5.0 µL of PCR product, 0.7µL of buffer enzyme, 1.0 µL of nuclease-free water (Qiagen, Germany), and 0.3 µL of enzyme restriction with a final total volume were 7.0 µL of RFLP mix.The restriction product was electrophoresed on 2.0% agarose gel using 100 Volt for 32 minutes, immersed in a 0.5X TBE solution, and then visualized using a UV Transilluminator.The size of the DNA bands that emerged was ascertained by comparing them with a 100 bp DNA ladder to determine the size of the DNA band and the MSTN gene's genotype.

Data analysis
The genotype frequency represents the proportion of a specific genotype within a population, obtained by comparing the occurrence of that genotype across populations.On the other hand, the allele frequency indicates the proportion of a specific allele concerning the total alleles present within a population [13].The data analysis of genotype frequencies, allele frequencies, heterozygosity value, and PIC (polymorphism information content) were calculated using Popgene32 software.
The following formulas were used to calculate the genotype and allele frequencies [14]: The following formulas were used to estimate the observed and expected heterozygosity [15]:

Polymorphism of the MSTN gene
The identification of the MSTN gene diversity in several beef cattle breeds in Indonesia was performed using the PCR-RFLP method with two restriction enzymes, HaeIII (Biolabs Inc., New England), which cleaves explicitly the (5'-GGCC- Genotype frequencies, allele frequencies, heterozygosity value, and the polymorphic information content (PIC) of the MSTN gene of Madura, PO, Bali, Simmental, and Limousin cattle are shown in Table 1 for the HaeIII result and Table 2 for the AluI result.Table 1 shows that the GG genotype has the highest frequency among all beef cattle breeds.The HaeIII enzyme only cleavage at target mutation in Madura and PO cattle that resulted in CG and GG genotypes, but not in Bali, Simmental, and Limousin cattle, which were monomorphic and only had a 100% of the GG genotype.The analysis results for the AluI enzyme in Table 2 showed that the MSTN gene in Madura, PO, and Bali cattle was polymorphic but monomorphic in Simmental and Limousin.The genetic diversity of a population can be considered polymorphic when none of its alleles exceeds 99% or 0.99 [16].The genotype frequencies of the MSTN│HaeIII gene in both Madura and PO cattle show that the GG genotype had the highest frequency, followed by the CG genotype.Both Madura and PO cattle did not show any of the CC genotypes.As a result, the frequency of the G allele in both cattle became much higher than that of the C allele.However, no diversity was observed in Bali, Simmental, and Limousin cattle that only had the GG genotype.Madura and PO cattle are polymorphic because both cattle breeds resulted from crossbreeding between Bali cattle (Bos javanicus) as native cattle in Indonesia and Zebu cattle (Bos indicus) imported from the Indian region.The PO cattle breed is the result of crossbreeding between white-colored Sumba Ongole (SO) cattle and brown-colored Bali cattle (Bos javanicus) since 1930 [17].The SO cattle are also formed from the pure mating of ongole or zebu cattle imported from India since 1914 in the breeding station at Sumba island and have been continuously bred and well adapted on the Sumba island [18].A similar occurrence also happened to Madura cattle, which is the result of a crossbreeding between Bali cattle (Bos javanicus) and Ongole cattle (Bos indicus) when people from India came to Indonesia 1500 years ago and had been continuously breeding on the Madura island [19].[20] that reported three genotypes in the PO cattle population using sequencing techniques: CC, CG, and GG.However, similar results were obtained regarding the genetic diversity of the MSTN gene in Madura cattle, which only showed the CG and GG genotypes.Some information could indicate that crossbreeding between cattle breeds led to heterosis or segregation effects, forming other genotype variations.If an adequate sample size were available, the CC genotype should be present in Madura and PO cattle.This anomaly could have been caused by several reasons, such as a limited sample size that might not adequately represent the population, genetic drift, or the possibility of eliminating cattle with the CC genotype [21].
The result of genotype frequencies and allele frequencies of the MSTN│AluI gene is shown in Table 2.The AA genotype in Madura cattle had the highest frequency, followed by the AG genotype.PO cattle also had a high frequency of GG genotypes but had a low AG genotype and very low frequencies for the AA genotype.In Bali cattle, the AA and GG genotypes showed higher frequencies compared to IOP Publishing doi:10.1088/1755-1315/1292/1/0120036 Madura and PO cattle, but they had the lowest frequency of the AG genotype.The high frequency of the AA genotype in PO, Madura, and Bali cattle resulted in the frequency of the A allele becoming higher than the frequency of the G allele.Breeding programs, such as selection, can be implemented when the population is polymorphic.Crossbreeding can be performed when it is uniform or monomorphic [13].  2 indicate that the HaeIII and AluI enzymes used to determine polymorphism of the MSTN gene in exon 1 only resulted in the GG genotype.These results followed other studies in which the MSTN gene's genetic diversity in exon 1 of Simmental cattle shows a monomorphic result [22].The genetic diversity of the MSTN gene, as reported in previous studies, also indicated that the MSTN gene exon 1 of Limousin cattle is monomorphic [23].
Genetic diversity is one of the analyses used as a reference for breeding programs.The diversity of the MSTN gene in different beef cattle breeds in Indonesia can serve as valuable data for confirming the validity of genetic markers.The validity can be achieved by conducting additional examinations, such as investigating the association between MSTN gene genotypes and various economically significant productivity traits.Therefore, further studies need to be conducted to investigate the association between the genetic diversity of MSTN genes generated in this research and productivity parameters (birth weight, weaning weight, carcass percentage, and muscle characteristics) in beef cattle.Eventually, the genetic diversity of MSTN genotypes can serve as a basis for marker-based breeding programs in Indonesia, utilizing genetic markers for selection and directed mating mechanisms.

Conclusion
The SNPc.111G>C of the MSTN│HaeIII gene resulted in polymorphism in Madura and PO cattle while monomorphic in Bali, Simmental, and Limousin cattle.The SNP267G>A of the MSTN gene can be cleaved by the AluI enzyme, leading to polymorphism in Madura, PO, and Bali cattle, while monomorphic in Simmental and Limousin cattle.This research provided information for further analysis, such as an association of the MSTN gene with some economic traits in Indonesian local beef cattle.
; Nii = total of genotype ii; nij = total of genotype ij; and N = total sample size.
the observed individuals; nij = number of heterozygote individuals; q = number of alleles; and Xi = frequency allele.

3. Result and Discussion 3 . 1
Amplification of MSTN geneThe forward and reverse primer used in this study is a primer designed based on the available MSTN gene sequence information in GenBank (www.ncbi.nlm.nih.gov) with accession number AY794986.1 resulting in a DNA sequence with a length of 608 base pairs (bp).The results of the MSTN gene sequence amplification in Madura, PO, Bali, Simmental, and Limousin cattle in this research are presented in Figure2.Based on the visualization of the MSTN gene amplification in this study, it is known that the MSTN gene in several beef cattle breeds in Indonesia was successfully amplified with the intended 608 bp target size.
3') base sequence and AluI that cleaves the (5'-AGCT-3') sequence.The results of the DNA sequence cleaving the MSTN gene in several beef cattle breeds in Indonesia are shown in Figures 3 (HaeIII) and 4(AluI).The HaeIII enzyme cutting against SNPc.111G>Cresulted in two alleles (C and G) and two genotypes, CG and GG (Figure 3).The GG genotype had two bands of 435 bp and 173 bp lengths.The CG genotype had three bands with 435 bp, 380 bp, 173, and 55 bp lengths.The target of SNPc.267G>A in the MSTN gene sequence was successfully cleaved by the AluI enzyme, resulting in 3 genotypes, GG (322 bp and 199 bp), AG (322 bp, 261 bp, 199 bp), and AA (322 bp and 261 bp.The DNA bands with lengths below 100 bp may not be visible because they are too short to be seen.

Table 1 .
Genotype and allele frequencies, heterozygosity, and PIC for SNPc.111G>CHe= expected heterozygosity; Ho= observed heterozygosity The diversity of the MSTN│HaeIII gene of the PO cattle population in this study had different results from previous research

Table 2 .
Genotype and allele frequencies, heterozygosity, and PIC for SNPc.267G>AN=number of samples; He= expected heterozygosity; Ho= observed heterozygosity The MSTN gene in Simmental and Limousin cattle in this research was monomorphic.The results in Table1 and Table