Open Veterinary Journal, (2026), Vol. 16(4): 2281-2287

Research Article

10.5455/OVJ.2026.v16.i4.28

Preliminary study of genetic variation in the CYP26B1 gene coding region in Bali cattle bulls

Nelyta Mulyati¹, Ronny Rachman Noor² and Jakaria Jakaria^2*

¹Graduate School of Animal Production and Technology, Faculty of Animal Science, IPB University (Bogor Agricultural University), Bogor, Indonesia

²Department of Animal Production and Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia

*Corresponding Author: Jakaria Jakaria. Department of Animal Production and Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia. Email: jakaria [at] apps.ipb.ac.id

Submitted: 13/11/2025 Revised: 03/03/2026 Accepted: 16/03/2026 Published: 30/04/2026

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ABSTRACT

Background: The cytochrome P450 family 26 subfamily B polypeptide 1 (CYP26B1) gene plays a crucial role in the regulation of retinoic acid metabolism, which is involved in various physiological processes in cattle, including reproduction and development. Therefore, studying genetic variation in the CYP26B1 gene can provide valuable baseline information for understanding its molecular diversity in cattle.

Aim: This study aimed to identify single-nucleotide polymorphisms (SNPs) and assess the genetic diversity of CYP26B1 in Bali bulls.

Methods: Blood samples were collected from 10 Bali cattle bulls at the Artificial Insemination Center in Singosari, Malang, East Java, Indonesia. The coding region of CYP26B1 was amplified and sequenced. SNP identification and sequence analyses were performed using the MEGA software version 12. Allele and genotype frequencies, observed and expected heterozygosity (Ho and He), and Hardy–Weinberg equilibrium were evaluated using a chi-square (χ²) test.

Results: Six SNPs were identified within the 1.539-bp coding region of the CYP26B1 gene, namely c.528 C>T, c.624 G>A, c.948 G>A, c.1005 C>T, c.1098 G>T, and c.1125 G>A. Five SNPs showed an allele frequency of 0.95/0.05, while SNP c.1125 G>A showed an allele frequency of 0.40/0.60. All SNPs identified were synonymous and did not result in amino acid substitutions. None of the SNPs within this sample deviated from the Hardy–Weinberg equilibrium (χ²=1.7–3.2; p > 0.05).

Conclusion: The SNPs identified in Bali bulls indicate low-to-moderate genetic variation among the individuals sampled. These findings provide preliminary molecular information that may serve as a reference for future studies using larger sample sizes and functional approaches to clarify the potential relevance of CYP26B1 variation.

Keywords: Bali cattle, CYP26B1 gene, Candidate gene, Genetic diversity, SNP.

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Introduction

Indonesia has abundant livestock genetic resources, with Bali cattle being one of the most important indigenous beef breeds. Bali cattle are commonly found not only on Bali island but also across eastern Indonesia, especially in South Sulawesi, East Nusa Tenggara, and West Nusa Tenggara. These cattle descended from the domestication of banteng (Bos javanicus), which occurred over thousands of years in Indonesia and Southeast Asia. Bali cattle are more closely related to banteng than to Bos taurus or Bos indicus (Wang et al., 2025).

The national cattle population in Indonesia remains substantial, with approximately 11.75 million heads, with Bali cattle accounting for approximately 27% of the total (Badan Pusat Statistik, 2024). This makes Bali cattle a key component of the country’s beef production. They are known for several beneficial traits, including good adaptability to tropical environments and relatively high fertility rates, which are reported to be around 83% and 75%, respectively, under normal and more challenging environments, such as the Timor region (Hariyono et al., 2025). Despite these advantages, increasing productivity and ensuring long-term sustainability of Bali cattle remain challenges, emphasizing the need for ongoing conservation and genetic improvement efforts (Warmadewi et al., 2017; Sari et al., 2022).

Efforts to conserve and improve Bali cattle can be conducted using both traditional and molecular techniques. Molecular genetics has become increasingly vital in supporting breeding programs, especially by identifying genetic variation in candidate genes linked to economic traits. One such candidate gene is cytochrome P450 family 26 subfamily B polypeptide 1 (CYP26B1), which plays a crucial role in retinoic acid (RA) metabolism and is involved in various physiological processes, including reproduction, growth, and health (Sejian et al., 2018; Isoherranen and Zhong, 2019; Zhao et al., 2024).

Molecular methods, such as DNA sequencing and single-nucleotide polymorphism (SNP) analysis, allow for more accurate characterization of genetic variation than phenotypic selection alone. These methods can help improve selection strategies while also preserving genetic diversity and reducing the risk of genetic erosion in indigenous cattle breeds (Husien et al., 2024).

However, information about genetic variation in the coding region of the CYP26B1 gene in Bali cattle is limited. To our knowledge, no previous study has reported SNPs within the coding region of the CYP26B1 gene in Bali bulls using DNA sequencing methods. Therefore, this study aimed to identify and analyze genetic variation in the coding region of the CYP26B1 gene in Bali bulls sampled from the AIC in Singosari, Malang, East Java, Indonesia.

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Materials and Methods

Animals and sample collection

All Bali cattle used in this study were male Bali bulls raised at a single AI Center in Singosari, Malang, East Java, Indonesia. The cattle were managed according to standard operating procedures, including forage and concentrate feeding. Ten Bali bulls were sampled, and blood was collected from the jugular vein by a licensed veterinarian. Because all samples came from the same AI center, the observed genetic variation might reflect a shared genetic background, and potential inbreeding bias should be considered when analyzing the results.

DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing analysis

DNA extraction was performed using the Geneaid Genomic DNA Mini Kit, following the five-step protocol: sample preparation, cell lysis, DNA binding, washing, and DNA elution. Primer3 (https://primer3.ut.ee/) and Primer Stats (https://www.bioinformatics.org/sms2/pcrprimer_stats.html) were used to design the primers for amplifying the coding region of the CYP26B1 gene (accession code ENSBTAG00000012212). Table 1 shows the target fragment, sequence, product length, and primer annealing temperature.

Table 1. Primer sequences used for PCR amplification of the CYP26B1 gene coding region (exons 1–6) in Bali bulls.

PCR amplification was conducted in a total reaction volume of 25 µl, containing 1 µl of genomic DNA template, 0.3 µl of each forward and reverse primer, 12.5 µl of Redmix PCR Master Mix, and nuclease-free water. Amplification was performed using a thermal cycler with an initial denaturation at 95°C for 1 minute, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing for 15 seconds at exon-specific temperatures (58.0°C for exon 1; 59.6°C for exons 2 and 4; and 61.1°C for exons 3, 5, and 6) and extension at 70°C for 10 seconds. A final extension step was performed at 72°C for 1 minute. The PCR products were separated by 1% agarose gel electrophoresis and visualized under ultraviolet light after staining. The amplified fragments corresponding to each coding region segment were then sequenced in the forward direction by a commercial sequencing service (1stBASE, Selangor, Malaysia) through PT Genetika Science.

Analysis of the CYP26B1 gene sequence data

The sequencing data were aligned using the ClustalW algorithm implemented in MEGA version 12 (Hall, 2011; Kumar et al., 2024) to analyze genetic variation within the CYP26B1 gene. Allele frequencies, genotype frequencies, observed heterozygosity (Ho), and expected heterozygosity (He) were then calculated. Phylogenetic analysis was performed based on the CYP26B1 coding region sequences using the neighbor-joining method with 1,000 bootstrap replicates in MEGA12. For comparison, CYP26B1 coding sequences from other bovine species, including B. javanicus, B. indicus, B. taurus, and related Bos species, were obtained from the GenBank database. The CYP26B1 coding sequences assembled in this study have been submitted to the GenBank database under accession number PX780313. In addition, Ramachandran plots and three-dimensional protein structure models of CYP26B1 were generated using the SWISS-MODEL server (https://www.expasy.org/).

Ethical approval

This study was approved by the Animal Ethics Committee of IPB University (Bogor Agricultural University), with approval number 318-2025 IPB.

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Results

Amplification of CYP26B1

The CYP26B1 gene was successfully amplified in this study using primers targeting the coding region in Bali bulls (Fig. 1). Fig. 1 shows the results of agarose gel electrophoresis of the six amplified coding fragments, each shown in separate gels. Clear and specific DNA bands corresponding to the expected fragment sizes were observed, indicating successful target region amplification. The amplified CYP26B1 coding region consisted of six fragments: exon 1 (514 bp), exon 2 (339 bp), exon 3 (382 bp), exon 4 (309 bp), exon 5 (412 bp), and exon 6 (453 bp). The successful amplification of all fragments indicated that the genomic DNA template was of adequate quality, with concentrations ranging from 20 to 50 pg/µl and purity values between 1.8 and 2.0.

Fig. 1. Agarose gel electrophoresis of PCR products amplifying the CYP26B1 gene coding region (exons 1–6) in Bali bulls. M indicates a 100 bp DNA ladder, and lanes 1–5 represent representative Bali bull samples showing successful target fragment amplification.

Determination of SNPs and CYP26B1 diversity

Genetic sequencing analysis of the CYP26B1 coding region in Bali bulls identified six SNPs (Table 2 and Fig. 2). For SNP c.624 G>A, only homozygous genotypes were identified, and no heterozygous genotype was observed among the individuals analyzed. The analyzed CYP26B1 coding sequence was 1.539 bp in length, spanning from the ATG to the TAA and encoding a protein of 512 amino acids. All SNPs detected were synonymous substitutions and did not result in amino acid changes. Structural validation of the predicted CYP26B1 protein indicated good model quality. The Ramachandran plot indicated that 92.9% of residues (418 residues) were located in the most favored regions, 6.9% (31 residues) in the allowed regions, and only 0.2% (1 residue) in the disallowed region, confirming the three-dimensional protein model’s reliability (Fig. 3). Figure 4 shows the phylogenetic analysis based on the CYP26B1 coding region sequences from Bali bulls and reference sequences retrieved from GenBank. The resulting phylogenetic tree illustrates the clustering pattern of Bali bull sequences relative to other Bos species, reflecting the genetic relationships inferred from the similarity.

Table 2. SNPs and genetic diversity parameters of the CYP26B1 gene coding region in Bali bulls.

Fig. 2. Identification of SNPs in the CYP26B1 gene coding region.

Fig. 3. Ramachandran plot (A) and three-dimensional structure (B) of CYP26B1 in Bali cattle. The three-dimensional structure was generated using the SWISS-Model (https://swissmodel.expasy.org/).

Fig. 4. The phylogenetic tree was constructed based on the coding region sequences of the CYP26B1 gene from 10 Bali bulls and reference sequences retrieved from GenBank, including B. javanicus (n=2), B. indicus (n=1), B. taurus (n=1), B. mutus (n=1), B. bison (n=1), B. bubalis (n=1), and B. kerabau (n=1). The bootstrap values were calculated from 1,000 replicates and are indicated at the nodes. The scale bar represents 0.0010 nucleotide substitutions per site.

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Discussion

The high-quality genomic DNA obtained in this study enabled efficient and consistent amplification of the CYP26B1 coding region, ensuring the reliability of subsequent molecular analyses. The primers designed for CYP26B1 showed high specificity and amplification efficiency, allowing for the accurate identification of SNPs and the assessment of genetic variation in Bali bulls. These results suggest that the PCR strategy used in this study was suitable for detecting sequence variation and provides a solid methodological foundation for future genetic research, including exploratory studies related to marker-assisted selection (Delghandi et al., 2022; Dairoh et al., 2023; Pertiwi et al., 2024).

All SNPs detected were synonymous mutations and did not cause amino acid substitutions or alter the predicted protein sequence (Bailey et al., 2021). Although synonymous mutations are typically considered functionally neutral, their possible effects on mRNA stability or translation efficiency were not assessed in this study and thus remain speculative. Additional functional analyses are needed to determine the biological significance of these synonymous variants (Kristofich et al., 2018; Shen et al., 2022).

The genetic variation found in the CYP26B1 coding region offers baseline information that could be helpful for future research on this gene in Bali cattle. Although Bali cattle are well known for their adaptability to tropical environments, this study did not evaluate the gene expression levels or functional traits related to the identified SNPs. Therefore, no definitive conclusions can be made regarding how CYP26B1 variation affects physiological or production traits. The data from this study may serve as a foundation for future research exploring links between CYP26B1 polymorphisms and economically important traits, especially when supported by larger sample sizes, diverse populations, and functional validation studies (Sudrajad et al., 2022).

Phylogenetic analysis based on the CYP26B1 coding region sequences showed that Bali cattle (Bos javanicus) clustered separately from B. taurus and B. indicus, as well as from more distantly related species such as Bubalus bubalis, Bubalus kerabau, Bison bison, and Bos mutus. The clustering pattern observed in the phylogenetic tree reflects evolutionary relationships inferred from sequence similarity within the CYP26B1 coding region and is consistent with previously reported phylogenetic relationships among these species. Nevertheless, the observed phylogenetic structure is based solely on sequence similarity and provides no direct evidence of functional differences in RA metabolism or specific physiological adaptations.

Greater genetic distances were observed between Bali cattle and B. taurus, which may reflect differences in evolutionary history and domestication pathways rather than specific adaptive traits. Bali cattle retain genetic characteristics associated with their banteng ancestry, whereas B. taurus cattle have undergone distinct selection processes in temperate regions. Similarly, buffalo and bison species appeared to be more distantly related to Bali cattle in the phylogenetic analysis, consistent with their divergence within the Bovidae family and their adaptation to different ecological niches (Warman et al., 2024).

Overall, the phylogenetic analysis based on the CYP26B1 coding region offers descriptive insights into the genetic relationships among cattle and related species. Although these findings help improve the understanding of the evolutionary background of the CYP26B1 gene, further studies incorporating functional assays, gene expression analyses, and association studies are necessary to clarify the role of CYP26B1 polymorphisms in economically significant traits. Such comprehensive approaches will be crucial before considering CYP26B1 as a candidate gene in molecular-based cattle selection programs.

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Conclusion

This study identified six SNPs within the 1.539-bp coding region of the CYP26B1 gene in Bali bulls. All identified SNPs were synonymous and did not lead to amino acid substitutions, indicating low-to-moderate genetic variation among the studied individuals. These findings provide initial molecular insights into the CYP26B1 gene in cattle in Bali. However, further research involving larger sample sizes, diverse genetic backgrounds, and functional or association analyses is necessary to clarify the biological significance of these synonymous SNPs and their potential links to economically important traits.

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Acknowledgments

The authors would like to thank the AI Center in Singosari, Malang, East Java, Indonesia, and the Animal Molecular Genetics Laboratory at IPB University in Bogor, West Java, Indonesia, for their assistance.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This research is funded by the Ministry of Higher Education, Science, and Technology, Indonesia, Decree Number: 006/C3/DT.05.00/PL/2025 dated May 28, 2025, and Agreement/Contract Number: 23297/IT3.D10/PT.01.03/P/B/2025.

Author’s contributions

All authors have contributed to this research. NM handled data collection and manuscript drafting. JK and RRN critically revised the manuscript. All authors have read and approved the final version of the manuscript.

Data availability

All data are included in the revised manuscript.

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