The genetic polymorphism of c.-1221A>C and discovery of novel SNPs in the promoter region of Mitochondrial Transcription Factor A (TFAM) gene in Bali Cattle

The expression of the mitochondrial transcription factor A (TFAM) gene has a marked impact on mitochondrial biogenesis. It is suggested to play a critical role in lipogenesis, including the deposition of marbling fat in cattle. Bali cattle (Bos javanicus) are known for their lean meat but exhibit poor marbling scores. This study aimed to investigate the polymorphism of c.-1221A>C and novel SNPs in the promoter region of the TFAM gene in Bali cattle. A total of 245 Bali cattle individuals were included in this study. Three Bali, three Wagyu, and one Limousin cattle were used for sequencing analysis. Genomic DNA was extracted either from blood or sperm for genotyping and variant analysis. The c.-1221A>C SNP was genotyped by PCR-RFLP, and the novel SNPs of the TFAM gene promoter region were identified by sequencing analysis. Our results revealed that the c.-1221A>C SNP was found to be monomorphic, with the presence of a fixed A allele, making it unsuitable for further association study in Bali cattle. However, the study successfully identified four novel SNPs and one novel DNP found in the promoter region of the TFAM gene. Notably, one double nucleotide polymorphism (DNP) of g.[45690945C>T;45690946A>G] was unique in Bali cattle compared to other breeds. In addition, only the c.-911T>A SNP was found heterozygous within the Bali cattle samples. These SNPs provide valuable insights into the genetic diversity of the TFAM gene in Bali cattle and serve as a basis for future investigations on the distinctiveness of Bali cattle, especially in the efforts of enhancing the quality of Bali beef.


Introduction
The importance of consumer expectations for high-quality beef meat has become increasingly evident in modern lifestyles [1].Meat quality assessment involves intrinsic and extrinsic factors [2].Intrinsic quality refers to the product's characteristics, while extrinsic quality includes animal health and welfare, environmental impact, and price considerations.A key determinant of intrinsic quality in beef is intramuscular fat deposition, commonly known as marbling.Marbling refers to white spots or lines of fat distributed within the muscle fibers of adipose tissue [3].The amount of marbling fat in beef significantly influences its flavour, aroma, and texture.The presence of fat contributes to a more prosperous and savoury flavour while improving the tenderness of the meat.Unfortunately, local Indonesian cattle such as Bali cattle (Bos javanicus) exhibit low marbling scores, resulting in relatively more hard meat compared to exotic beef breeds such as Wagyu cattle [4][5][6][7].
Genetic selection and feed manipulation are viable approaches to improve beef quality by establishing desirable traits, such as marbling, in the genetic makeup.Identifying genetic diversity in genes that directly influence marbling is critical for conducting selection activities at the DNA level.Mitochondrial transcription factor A (TFAM) is a member of the High Mobility Group Box (HMGB) subfamily of DNA-binding proteins that plays a critical role as a transcription factor for mitochondrial DNA (mtDNA) by binding to the mtDNA promoter [8].TFAM maintains mtDNA integrity by regulating mtDNA copy number and transcription [9,10].Reduced levels of TFAM will reduce mtDNA copies, which impairs mitochondrial ATP production [11].Furthermore, TFAM expression has been shown to affect mitochondrial biogenesis during in vitro differentiation of mouse brown fat cells [12].Although the regulatory pathways are not fully understood, the TFAM gene is thought to play an essential role in bovine lipogenesis due to its involvement in mitochondrial biogenesis [13].The TFAM gene is likely associated with the amount and function of mitochondria, which serve as sites for lipid metabolism [14].While the regulation of lipid metabolism differs between monogastric (non-ruminant) and polygastric (ruminant) animals [15][16][17] demonstrated the role of the TFAM gene in gene networks related to subcutaneous fat deposition, establishing a link between the TFAM gene and lipid metabolism in cattle.
The investigation of TFAM gene diversity in cattle intrigues due to its strong association with carcass traits such as marbling score and subcutaneous fat thickness [13], as well as reproductive traits such as embryo development [18] and occurrence of repeat mating [19].The TFAM gene expression was also significantly associated with feed efficiency in Nellore cattle [20].In relation to meat quality traits, [13] discovered two highly significant SNPs within the TFAM gene that correlated with marbling score and subcutaneous fat thickness in an F2 Wagyu X Limousin crossbred cattle population.One of these SNPs was c.-1221A>C, located in the promoter region of the TFAM gene.This SNP had a strong correlated to marbling score (P=0.015) and subcutaneous fat thickness (P=0.02), with the C allele tending to be associated with higher marbling scores and thicker subcutaneous fat than the A allele.
While Bali cattle are known to have the advantage of lower fat content compared to other local and exotic cattle [5], they tend to have lower marbling scores [21].Identification of additional candidate genes, including the TFAM gene, is necessary to improve the quality of Bali beef, especially to achieve higher marbling scores [22].Therefore, this study aimed to investigate the diversity of the TFAM gene, particularly the SNP c.-1221A>C, and to explore other mutations in the promoter region of the TFAM gene as potential alternative genetic markers in the Bali cattle population.

Sample collection
A total of 245 individuals of Bali cattle were used and collected for blood samples (200) and sperm samples (45).The blood samples were obtained from 60 individuals in Enrekang Regency, 68 individuals in Barru Regency, both located in South Sulawesi Province and 72 individuals from Nusa Penida Island in Bali Province.Meanwhile, the sperm samples were collected from 35 Bali bulls raised by the National Artificial Insemination Center of Singosari, East Java Province (NAIC-Singosari), and 11 Bali bulls raised by the Regional Artificial Insemination Center of Banyumulek, West Nusa Tenggara Province (RAIC-Banyumulek).This research was conducted by adhering to ethical guidelines for the use of animals in experiments, with efforts made to minimize any discomfort or harm to the animals involved.
For blood sample collection, vacutainer tubes containing K3 EDTA were used, while the sperm samples were obtained from frozen semen straws.Genomic DNA was extracted from the blood samples using the Genomic DNA mini kit (Genaid Biotech Ltd., Taiwan), and from the sperm samples using the gSYNC DNA extraction kit (Genaid Biotech Ltd., Taiwan), following the manufacturer protocol.

PCR RFLP genotyping
A pair of primer sequences designed by [13] was used in this study (Table 1).The primer sequences were confirmed using the Primer-BLAST program at NCBI, and the corresponding products were located in the Bovine Genome Database with the Refseq ID number of NC_037355.1 which harbors the TFAM gene (Gene ID: 510059) with a length of 16440 bp spanning from the 4567338 base to 45689820 base.The PCR reaction was in a total volume of 10 µL, consisting of 10-12 ng DNA template, 5 µL MyTaq™ HS Red Mix (Meridian Bioscience, USA), 0.2 µL each of the forward and reverse primers, and 3.6 µL nuclease-free.The PCR program began with a pre-denaturation step at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 57°C for 15 seconds, and initial extension at 72°C for 24 seconds.The PCR cycle was completed with a final extension step at 72°C for 5 minutes.The PCR products were electrophoresed on a 1% agarose gel containing GelRed nucleic acid gel stain (Biotium, USA) at a dilution of 1:10000.Electrophoresis was conducted using Mupid-exU (Japan) at 100 volts for 30 minutes.Subsequently, the gel was visualized using the GBOX gel documentation system (Syngene, UK).
The locus of interest for TFAM gene polymorphism was the A/C base substitution at position c.-1221 [13].Genotyping was performed using the PCR-RFLP method with the restriction enzyme HaeIII (Thermo Scientific, Lithuania), which recognizes and cuts at the 5'-GG↓CC-3' restriction site.The mixture composition and the RFLP procedure were adjusted according to the manufacturer's instructions.The RFLP products were electrophoresed on a 2.5% agarose gel containing GelRed nucleic acid gel stain (Biotium, USA) at a dilution of 1:10000.Electrophoresis was conducted using Mupid-exU (Japan) at 100 volts for 40 minutes.Subsequently, the gel was visualized using the GBOX gel documentation system (Syngene, UK).The genotype of individual cattle was determined by analyzing the banding pattern according to [13].Band sizes were determined using a 100 bp DNA marker (Thermo Scientific, Lithuania).If the genotype is AA, three bands will be visible (152, 187, and 462 bp), while if the genotype is CC, four bands will be visible (83, 104, 152, and 462 bp) and genotype AC is a combination of both (83, 104, 152, 187 and 462).

Sequencing and data analysis
The reference bovine genome (RefSeq NC_037355.1)and the sequence data from [13] were utilized to determine the position of the c.-1221A>C SNP and to identify potential novel variants in the Bali cattle population and other breeds.For this purpose, three randomly selected DNA samples from Bali cattle, one from Limousin cattle, and three from Wagyu cattle were subjected to bidirectional sequencing.The sequencing process was conducted using an ABI PRISM 96-capillary 3730xl DNA Analyzer (Applied Biosystems, USA) through the services provided by First Base sequencing.
To identify novel single nucleotide polymorphisms (SNPs), sequence analysis of the obtained PCR products was conducted.These sequences were aligned with the sequences of F2 Wagyu X Limousin cattle from a study of [13], as well as with the Refseq NC_037355.1 from NCBI, Limousin cattle, and Wagyu cattle sequences.The alignment and comparison of sequences were performed by using the BLAST program available at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi),Bioedit software [23], and MEGA11 for sequence alignment analysis [24].

PCR product
The successful amplification of a specific fragment of the TFAM gene with an approximate size of 801 bp [13] was confirmed in this study (Figure 1).To determine the genomic position of the PCR-generated DNA fragment, a BLAST search was conducted against the Refseq NC_037355.1 genome.The results indicated that the fragment spans the promoter region of the TFAM gene, specifically located at bases 45690192 to 45690992 of the genome.The target SNP of interest, c.-1221A>C, corresponds to this fragment's specific base position of 45690757 (g.45690757A>C) (Figure 2).base pairs.The specific target SNP is located at position 45690757 (A>C), corresponding to position c.-1221A>C [13], as highlighted in green shading.It's important to note that the TFAM gene sequence in the NCBI genome database (RefSeq: NC_037355.1) is oriented in the complement or negative strand direction, while the primer sequences and PCR products are read in the reverse direction.

Novel SNPs in the promoter region of the TFAM gene
The TFAM gene promoter region was sequenced in both forward and reverse directions using primer pairs designed by [13].To ensure accuracy, the resulting sequences were assembled and analyzed using Bioedit software to correct any potential bases that might have been miscalled during the sequencing process.The contig assembly from three Bali cattle samples yielded sequences of 773 bp, 774 bp, and 777 bp, representing approximately 96.5% to 97% of the expected 801 bp sequence.Subsequently, the contig sequence obtained from the Bali cattle samples underwent further sequence alignment analysis using MEGA11 software [24].
The nucleotide sequence alignment aimed to identify novel SNPs within the TFAM gene promoter region in Bali cattle.The results, as shown in Figure 4, revealed several variants.Specifically, we identified eight variants, five of which were novel (Table 3).Among these, three variants had been previously reported in the reference SNP cluster ID (rsID) c.-995T>C and the study by [13]

Discussion
The polymorphism of SNP c.-1221A>C in the TFAM gene in Bali cattle showed that the SNP was monomorphic in Bali cattle, with only the AA genotype and A allele observed in all samples.This indicates a lack of genetic diversity at the c.-1221A>C locus, suggesting a natural genotype exists in Bali cattle.The provision of genetic improvement programs and intensive selection for specific traits, such as meat quality traits, may occur mutations at this locus.The diversity of the c.-1221A>C SNP in the TFAM gene across different cattle breeds worldwide is still an area of ongoing research (Table 4).Previous studies have reported its polymorphism in cattle breeds known for high genetic marbling scores, such as F2 Wagyu X Limousin crossbred cattle [13].In the study by [13], the frequencies of AC, CC, and AA genotypes were reported as 0.49, 0.32, and 0.19, respectively.Additionally, the frequency of the C allele (0.56) was found to be higher than that of the A allele (0.44), indicating genetic variation associated with marbling scores.However, in the present study on Bali cattle, the A allele was fixed with a frequency of 1.00, suggesting its consistent presence in this breed.In another investigation by [26] on Nellore cattle (B.indicus), the frequency of the A allele was higher than that of the C allele, with values of 0.90 and 0.10, respectively.The allele distribution pattern observed in Nellore cattle differed from that of the overall Nellore cattle (control, selection, and traditional line) population, where the A allele was found at a very high frequency (0.84-0.94) [26].Similarly, [25] found that in B. taurus crossbred cattle, the highest allele frequency was in the C allele (0.58), which was higher than the A allele frequency (0.42).Notably, both studies by [13], and [25] utilized crossbred cattle.These variations in allele frequencies emphasize breed-specific differences at the c.-1221A>C locus.Further research incorporating purebred cattle of the B. taurus and B. indicus is necessary to elucidate the sources of allele flow and comprehend the genetic variation at the c.-1221A>C locus.From the current result (Table 3), the C allele comes from Wagyu cattle then to introduce genetic variation and potentially enhance marbling traits in the Bali cattle population, mating AA-genotyped male Bali cattle with AC-genotyped female Wagyu cattle can produce AC-genotyped offspring with a 50% probability.
The consistent presence of the A allele in Bali cattle indicates that the breed does not have a genetic propensity for high marbling scores.This finding is consistent with previous studies and reinforces the hypothesis proposed by [22] regarding the genetic predisposition of Bali cattle for high marbling scores.The absence of favourable alleles for high marbling scores in the identified candidate genes (TG5, AKIRIN2 and EDG1) supports the notion that Bali cattle did not have favourable alleles as high marbling cattle [22,27].In the TG5 gene, studies on Wagyu cattle by [28] showed a higher frequency of the TT genotype associated with high marbling scores, while in Bali cattle, only the CC genotype was observed [27].For the AKIRIN2 gene, the highest genotype frequency was identified as AG in Korean cattle [29], whereas the highest genotype observed in Bali cattle was GG.Similarly, for the EDG1 gene, the AG genotype had the highest frequency in Japanese Black cattle [30], whereas the Bali cattle had predominantly the AA genotype.
The discovery of a unique variant, NC_037355.1:g.[45690945C>T;45690946A>G], in Bali cattle compared to other cattle breeds, is a remarkable example of a multi-nucleotide polymorphism (MNP).MNPs occur when multi-consecutive nucleotides are altered relative to a reference sequence, MNPs occur when several consecutive nucleotides are altered relative to a reference sequence, two nucleotides (DNP), three nucleotides (TNP) [31].While single nucleotide polymorphisms (SNP) are recognized as an essential source of genetic variation in beef and dairy cattle [32], there has been limited research on MNPs in cattle has been conducted to date [33].In the human context, research has shown that MNPs are more likely to be associated with disease-causing mutations.
There are several reasons for this difference between SNPs and MNPs.Firstly, SNPs are often synonymous, meaning they change the nucleotide sequence but do not affect the amino acid sequence due to the redundancy in the genetic code [34].Secondly, while SNPs can at most cause a change in a single amino acid when an MNP is observed in the coding region, this might result in changes at two adjacent positions, leading to more dramatic effects on the encoded protein [31].This characteristic makes MNPs attractive candidates for functional analysis and identification of specific markers in livestock, including in Bali cattle.Furthermore, a heterozygous SNP observed at position c.-911T>A in the TFAM gene is also particularly interesting.This SNP has promising potential as a genetic marker in Bali cattle, especially in improving the quality of Bali beef, but it needs confirmation with more samples.

Conclusion
The c.-1221A>C SNP in the promoter region of the TFAM gene was found to be monomorphic in the Bali cattle population, indicated by the fixed presence of the A allele.Consequently, unsuitable for further association studies with any related traits in Bali cattle.However, our study successfully identified four novel SNPs and one novel DNP in cattle compared to other breeds, with one of them being heterozygous.Further studies with larger sample sizes and comprehensive phenotypic evaluations are warranted to validate the potential association between these novel SNPs, DNP, and beef meat quality traits, especially marbling, in Bali cattle.

Figure 2 .
Figure 2. The position of the PCR product derived from the TFAM gene, as per Refseq NC_037355.1.The blue lines represent the bases at the forward primer (45690992), while the red lines denote the bases at the reverse primer (45690192), encompassing the target product of 801base pairs.The specific target SNP is located at position 45690757 (A>C), corresponding to position c.-1221A>C[13], as highlighted in green shading.It's important to note that the TFAM gene sequence in the NCBI genome database (RefSeq: NC_037355.1) is oriented in the complement or negative strand direction, while the primer sequences and PCR products are read in the reverse direction.
, specifically at positions c.-1221A>C and c.-1213T>C.Notably, four out of the five novel SNP loci tended to be homozygous, namely c.-1427T>C, c.-1385G>C, c.-1121C>A, and c.-911T>A.Furthermore, we observed a unique variant in Bali cattle compared to other breeds, where two bases changed simultaneously at positions c.-1409C>T and c.-1410A>G or NC_037355.1:g.[45690945C>T;45690946A>G].Additionally, one SNP was found to be heterozygous at c.-911T>A.

Figure 4 .
Figure 4.The alignment of the nucleotide sequences of the Bali cattle TFAM gene promoter region with those of the bovine genome assembly at NCBI (Hereford) (accession number NC_037355.1 and GeneID: 510059), F2 Wagyu x Limousin[13], Limousin, and Wagyu cattle.

Table 1 .
[13]ence, position, and length from primer pair for amplification of a specific fragment of TFAM gene promotor area based on Refseq NC_037355.1.Primer designed by[13]; ** Position based on Refseq NC_037355.1 on complement sequence *

Table 2 .
Genotype and allele frequencies of TFAM gene identified in Bali cattle from five different locations

Table 4 .
Comparison of TFAM gene allele frequencies among various cattle breeds