Polymorphism of ADIPOQ and EDG1 genes in Indonesian beef cattle

The ADIPOQ and EDG1 genes were responsible in intramuscular fat deposition and marbling scores. This study was aimed to identify polymorphism of indel g.81966364D>I in promoter region of ADIPOQ gene and SNP c.-312A>G in 5' UTR of EDG1 gene in Indonesian beef cattle. Blood samples were collected from 211 cattle, including Bali (44), Madura (20), Pesisir (18), Katingan (20), PO (22), Pasundan (20), SO (12), Brahman (20), Simmental (15) and Limousin (18). Polymorphism of ADIPOQ gene was analyzed using PCR and direct sequencing methods, whereas EDG1 gene was analyzed using PCR-RFLP (MscI enzyme) and direct sequencing methods. Results of genotyping indel g.81966364D>I was monomorphic (DD genotype). The SNP c.-312A>G was polymorphic (AA and AG genotype) in Madura, Pesisir, Pasundan, Brahman, and Limousine. The Frequencies of allele A and G were 0.95, 0.92, 0.98, 0.95, 0.94 and 0.05, 0.08, 0.02, 0.05, 0.06 respectively. The values of Ho and He were 0.05Polymorphism of ADIPOQ and EDG1 Genes in Indonesian Beef Cattle (S. Sutikno et al.) 323 0.17 and 0.05-0.15 respectively and in Hardy-Weinberg equilibrium (P>0.05). In Bali, Katingan, PO, SO and Simmental were monomorphic (GG genotype). In Bali cattle, two novel SNP candidates were found in position of c.-399C>T and c.-273C>G which were potential to be used as genetic markers of marbling score for Bali cattle. As result this study, it can be concluded that ADIPOQ gene was similar while EDG1 gene was different in Indonesian beef cattle. in addition, found two candidates potential SNP in


INTRODUCTION
Indonesia has a lot of animals genetic resources consisting beef cattle such as Bali, Madura, Aceh, Sumbawa, Pesisir, PO, Jabres, and Sumba Ongole (SO). Animals genetic resources were important to manage for increasing the farmer's income and welfare, leading to national food security as well as the vevelopment of security as a nation. The policies of management of the animal genetic resources referred to three approaches, those were: Pure-breeding and Conservation, Cross breeding, and the development of new breeds. Several of Indonesia beef cattle were potential to be developed into premium beef cattle.
The value of the economic traits in beef cattle included birth weight, weaning weight, and meat quality. Selection of these traits based on phenotype results in a slow genetic improvement. the potential alternative selection was the markerassisted selection (MAS). Selection of livestock based on genetic markers could result in more accurate, effective and efficient. Selection could be decided based on candidate genes that control those traits (Van Werf and Kinghorn, 2003).
Several potential gene candidates related to meat quality included adiponectin (ADIPOQ) gene and endothelial differentiation sphingolipid G-protein-coupled receptor 1 (EDG1) gene. The ADIPOQ gene played a role in the process of lipogenesis, fatty acid oxidation, homeostatic energy, insulin sensitivity, and glucose utilization (Choi et al., 2015;Kwon et al., 2016). While the EDG1 gene played a role in intramuscular fat deposition (marbling) (Sasaki et al., 2006). The ADIPOQ gene had a promoter region which was a region of DNA that initiates transcription of a particular gene (Gershon and Kadonaga, 2010;Ohler and Wassarman, 2010;Lenhard et al., 2012). While the EDG1 gene had a 5' untranslated region (UTR) which has been recognized for the importance of regression of gene expression at the posttranscriptional level by affecting the mRNA stability, localization, and translational efficiency (Mignone et al., 2002).
The promoter of ADIPOQ gene had been identified in some of the world's cattle such as Angus cattle (Morsci et al., 2006), Chinese local cattle (Zhang et al., 2013) and Hanwoo cattle (Kwon et al., 2016). The 5'UTR of EDG1 gene was widely studied in wagyu cattle (Sasaki et al., 2006;Yamada et al., 2008;Sukegawa et al., 2010). However, information polymorphism the promoter region of ADIPOQ gene and the 5' UTR of EDG1 gene had not been explored in Indonesian beef cattle. Thus, the objective of this study was to identify the variation in the ADIPOQ promoter region and 5' UTR of EDG1 gene in Indonesia beef cattle.

Samples
The reseach was conducted in laboratory of molecular genetics of livestock, Faculty of Animal Science, Bogor Agricultural University. Blood samples were obtained from ten representative cattle breeds including Bali, Madura, Pesisir, Katingan, PO, Pasundan, SO, Brahman, Simmental and Limousin (Table 1).

DNA Isolation and PCR amplification
Blood samples were collected from jugular vein and kept into vaccuntainer tube containing ethanol absolut as anticoagulant. Genomic DNA was isolated using phenol-cloroform metods described by (Sambrook and Russel, 2001). Based on the bovine sequence (GenBank accession number JQ775868) and the cow sequence (ensembl accession number ENSBTAG00000005990), two pairs of primers (F: 5'-GCAGCTCTACTTGGCATCC-3' and R: 5'-TGAATCAGTCGTCCTTACCC-3') and (F: 5'-CGCAGATCTTTCCTGGACAG-3' and R: 5'-TTCTGCCTCTGAAGACCTCC-3') were designed to amplify in promoter region of the ADIPOQ gene and 5'UTR of the EDG1 gene, respectively. The primers were designed using MEGA7 and online evaluated using PCR Primer Stats (www.bioinformatics.org) for reducing error. For both genes, the 15 μLPCR amplification mix contained 50 ng of DNA template, 1× promega green master mix (based on the protocol provided by manufacturer) and 5 pmol of each primer. The PCR protocol was 5 min at 95°C, followed by 35 cycles of 95°C for 20 s, annealing at 60°C for 30 s, 72°C for 30 s and a final extension at 72°C for 5 min. The expected amplified fragment size for ADIPOQ and EDG1 genes were about 265 (no insertion) or 331 bp (67 bp insertion) and 411 bp, respectively.

Genotyping and Sequencing
For the genotyping to determine insertion or deletion in promoter region of the ADIPOQ gene, 2% agarose gel (0.6 g of agarose was diluted in 30 ml of 0.5×TBE buffer) electrophoresis was conducted, and genotypes were determined based on banding patterns of DNA for length 265 bp as deletion and 331 bp as insertion. For the genotyping to determine singel nucleotide polymorphism (SNP) c.-312A>G in the 5'UTR of the EDG1 gene using PCR-RFLP method. PCR product was digested at 37°C for 2 h with restriction enzyme MscI (TGG↓CCA) and electrophoresed on a 2.0% agarose gel. Agarose gels were stained with florosafe and photographed under an ultraviolet light. For restriction, the reaction mix contained 1.0 μLendonuclease free H 2 O, 5 μLPCR product, 0.7 μL MscI buffer, and 0.3 μL(3 U) MscI restriction enzyme. 411 bp PCR fragments containing the SNP site were digested by MscI into 186 and 225 bp fragments at the A allele, but not at the G allele, so the AA homozygotes, the GG homozygotes, and the AG heterozygotes resulted in two bands (186 and 225 bp), one band (411 bp) and three bands (186, 225 and 411 bp) respectively.
Sequencing was done only for Bali and Limousin cattle for promoter region of the ADIPOQ and 5'UTR of the EDG1 genes that had different genotypes (2 samples/genotype) using forward primer. Samples for sequencing were sent to commercial laboratory service at First BASE Laboratories Sdn. Bhd. (Selangor, Malaysia) using ABI PRISM 96-capillary 3730xl DNA Analyzer (Applied Biosystems, USA).

Data Analyses
Frequency of Genotype and Allele. Frequency of genotype and allele were calculated based on Nei and Kumar (2000) formula with the following statistical model: χ ii = (n ii /N) for genotype frequency and χ i = (2n ii + Σn ij )/(2N) for allele frequency, where: χ ii = frequency of ii genotype; χ i = frequency of i allele; n ii = number of individuals with ii genotype; n ij = number of individuals with ij genotype; N = number of samples.  Nei and Kumar (2000) formula with the following statistical model: where: He = expected heterozygosity; χ i = frequency of alleles; q = number of alleles.
Hardy-Weinberg Equilibrium (HWE). HWE was calculated according Hartl and Clark (1997) formula with the following statistical model: where: χ 2 = HWE test; O = observed number of genotype; E = expected number of genotype. Degree of freedom (df) for HWE test was defined according to Allendorf et al. (2013) where: df = number of genotype probabilities -number of alleles.

Sequence Analysis
Data of ADIPOQ and EDG1 genes sequences were analyzed by FinchTV and Bioedit program (Hall, 2011). The determination of SNP (single nucleotide polymorphism) was identified using Molecular Evolutionary Genetics Analysis 5 (MEGA5) (Tamura et al., 2011).

Amplification and Polymorphism of ADIPOQ Gene
The promoter region of ADIPOQ gene was amplified successfully at annealing temperature 60°C for 30 seconds. PCR product was 265 bp as shown in Figure 1, confirming the sizes using agarose gel electrophoresis and sequencing analyses. The present analysis determined genotypes according to length of DNA banding patterns, showing that the length 265 bp was assigned to D alleles (Deletion). The present PCR analysis did not find any cattle with insertion (331 bp), whereas 211 samples possessed D alleles, showing that allele frequency for D was 1.0. That the promoter region of ADIPOQ gene was monomorphic, when a locus in a population found only one allele or if the most common allele was known to be a high frequency (more than 95% or 99%), the locus was considered as monomorphic (Nei and Kumar, 2000;Allendorf et al., 2013). Allele frequency of indel g.81966364D>I in promoter region of ADIPOQ gene had been reported in several studies in Bos taurus, Bos indicus and its crossbreeds cattle as presented in Table 2. SNP indel g.81966364D>I in promoter region of ADIPOQ gene had been reported under significant effects on heart girth and huckle-bone in three Chinese cattle breeds (Zhang et al., 2013), marbling scores in Hanwoo cattle (Choi et al. 2015;Kwon et al., 2016). The sequences from promoter region of ADIPOQ gene were aligned with a reference sequence from NCBI (JQ775868.1), indicating that no insertion was found in Indonesian beef cattle.

Amplification and Polymorphism of EDG1 Gene
The 5'UTR of EDG1 gene was amplified successfully at annealing temperature 60°C for 30 seconds. Genotyping by restriction enzim MscI resulted in two genotypes: AA (225 and 186 bp) and AG (411, 225 and 186 bp) as presented in Figure 2. The present RFLP analysis did not find any cattle with GG genotype. Genotyping analysis x on 211 Indonesia beef cattle revealed that allele frequency of c.-312A>G gene fragment was high for A allele (digested by MscI), while for G allele (not digested by MscI) was considerably low frequency (Table 3). additions in some cattle populations had only A alleles as in Bali,  The results of this study indicating SNP c.-312A>G EDG1 gene in Limousine, Pesisir, Brahman, Pasundan, and Madura were polymorphic because the allele frequency was obtained more than 0.01 (Nei and Kumar 2000;Allendroft et al., 2013). While for Bali, Simmental, PO, Katingan and SO were monomorphic due to the frequency of alleles obtained less than 0.01 (Nei and Kumar 2000;Allendroft et al., 2013). The highest frequency of allele A in samples was estimated to be due to selection and mating control managed by farmers. The selection conducted by the breeder was to maintain cattle with A allele rather than with G allele. According to Noor (2010), factors affecting gene frequency were selection, mutation, inbreeding, crossbreeding and genetic drift.
The EDG1 gene encoded 383 amino acids and was associated with the traits of intramuscular fat deposition (marbling) in Wagyu cattle (Sasaki et al., 2006). Mutations in c.-312A>G was associated with marbling in Japanese Black cattle. Cattle with G alleles had higher marbling scores than cattle with A allele (Yamada et al., 2008;Sukegawa et al., 2010). The frequency of EDG1 gene alleles in various cattle in the world is presented in Table 4.
The heterozygosity value was the mean percentage of individual heterozygot or the percentage of heterozygot individuals in the population (Nei and Kumar, 2000). Observed and expected heterozygocity (Ho and He, respectively) values indicated that diversities of Indonesian beef cattle were remarkably low. The values were 0.05-0.17 and 0.05-0.15 for Ho and He, respectively (Table 3). Table 3 also showed that Ho and He values among the cattle breeds in this experiment were statistically similar. This indicated gene frequency in each population was in equilibrium state as supported by Hardy-Weinberg test in this experiment (P>0.05). Yet, Bali, Simmental, PO, Katingan and SO were an exception in which the gene frequencies in this population were considerably not in equilibrium state based on the test (P<0.05).
In general, population of Indonesian beef cattle was in dynamic equilibrium, with exception for Bali, Simmental, PO, Katingan and SO cattle population. This discrepancy might be due to limited sample number in this experiment. As Allendrof et al. (2013) reported that population size was one of constraint in Hardy-Weinberg equilibrium status. Other constraints were random matting, the absence of mutation, the absence of selection as well as the absence of migration.

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J.Indonesian Trop.Anim.Agric. 43(4):323-332, December 2018 (Choi et al., 2015;Kwon et al., 2016). The large difference between Ho and He values could be an indicator of imbalance genotype in population (Tambasco et al., 2003). The population of animals was expressed in equilibrium if the genotype and allele frequencies were constant from generation to generation (Allendorf et al., 2013). Large populations would not change from one generation to another if there was no selection, migration, mutation, and genetic drift (Noor, 2008)

Sequence Analysis of SNP c.-312A>G
Sequences analysis on A and G allele polymorphism in Bali and Limousin samples used reference from GenBank (access code NW_003103868 region: 12578072-12578482) and Ensembl (access code ENSBTAG00000005990). The result of alignment verified nucleotide transition at positions 189 (A>G) from forward PCR product 411 bp or at the   thyroglobulin (TG5) gene (Anwar et al., 2017), and 5'UTR of EDG1 gen (this study). Therefore, Bali beef cattle could be potential to be developed into premium beef cattle.

CONCLUSION
The indel g.81966364D>I of ADIPOQ gene was monomorphic in Indonesian beef cattle. While the SNP c.-312A>G of EDG1 gene was polymorphic in Pesisir, Brahman, Pasundan, Madura and Limousine cattle. In Bali cattle, two new SNP candidates were found in position of c.-399C>T and c.-273C>G which were potential to be used as genetic markers of marbling score.