Open Veterinary Journal, (2025), Vol. 15(9): 4295-4300

Research Article

10.5455/OVJ.2025.v15.i9.36

Polymorphism of g.462C>G in the NR2E1/PstI gene and its relationship with Japanese Quail (Coturnix japonica) economic traits

Widya Pintaka Bayu Putra¹, Abdullah Baharun², Dela Ayu Lestari^3*, Athhar Manabi Diansyah⁴, Annisa Rahmi², Deden Dwi Sutisna², Ikhsan Qodri Pramartaa², Muhammad Gitar Ramadhan² and Muhammad Aulia Reza⁵

¹Research Center for Applied Zoology, National Research and Innovation Agency, Cibinong, Indonesia

²Department of Animal Science, Faculty of Agriculture, Universitas Djuanda, Bogor, Indonesia

³Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Indonesia

⁴Faculty of Animal Science, Hasanuddin University, , Makassar, Indonesia

⁵Department of Animal Production and Technology Science, Faculty of Animal Science, IPB University, Bogor, Indonesia

*Corresponding Author: Della Ayu Lestari. Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Indonesia. Email: delaayulestari [at] ymail.com

Submitted: 30/04/2025 Revised: 29/07/2025 Accepted: 11/08/2025 Published: 30/09/2025

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Abstract

Background: Japanese quails (Coturnix japonica) are a poultry species that is kept for meat and egg production. Genetic improvement in Japanese quails is important to increase quail farmers’ income. Molecular selection can be applied in Japanese quails to increase productivity traits.

Aim: This study aimed to determine the effects of coturnine NR2E1 gene polymorphism on slaughter weight, carcass weight (CW), and carcass percentage (CP).

Methods: Fifty female Japanese quails (Coturnix japonica) were used as experimental birds. The birds were maintained under an intensive management system. A method based on polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis was used to detect a g.462C>G mutation site on the NR2E1/PstI gene.

Results: The results showed that the genetic diversity in the mutation site under study was high, with a polymorphic informative content value of more than 0.30. In addition, three genotypes of CC, CG, and GG were observed in the birds under study, with allele frequencies of 0.42 (C) and 0.58 (G). According to the Chi-square (χ²) value, the genetic diversity in the studied birds was not under genetic equilibrium (χ²>5.99). Nonetheless, polymorphism in g.462C>G was not significantly associated with the economic traits of the studied birds. Nonetheless, the heterozygous birds had higher CW and CP traits than other genotypes.

Conclusion: The coturnine NR2E1/PstI gene was polymorphic with high genetic diversity. However, no significant association was found between the NR2E1/Pst1 gene polymorphism and economic traits for Japanese quails under study. In the future, in-depth research on the coturnine NR2E1 gene involving a large sample and different gene regions is important to accurately obtain the genetic marker for economic traits.

Keywords: Economic traits, NR2E1 gene, PCR-RFLP, Polymorphism, Japanese quails.

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Introduction

The Japanese quail (Coturnix japonica) is a poultry animal that is kept for egg and meat production. A total of 15,227,131 Japanese quail heads were recorded in Indonesia, with a large population (74.19%) found on Java Island (Statistics of Indonesia, 2022). In 2022, quail farmers in Indonesia were able to produce 22,015.48 tons of eggs and 1,038.18 tons of meat (Indonesian Ministry of Agriculture, 2023). Genetic improvement in Japanese quails is important to increase quail farmers’ income. Molecular selection can be applied in Japanese quails to increase productivity traits. The nuclear receptor subfamily 2 group E member 1 (NR2E1) gene or T-cell leukemia homeobox (TLX) is a candidate gene for genetic improvement in Japanese quails (Sasazaki et al., 2006). This gene plays an important role in neural development through the progressive regulation of the cell cycle in the NSC system (Song et al., 2015). In Japanese quail, the NR2E1 gene spreads along 14,710 bases on chromosome 3 with 9 exons (GenBank: NC_029518.1). Unfortunately, studies on this gene are limited. However, a polymorphism of the NR2E1/PstI gene was reported by Ahmed and Al-Barzinji (2020) and Lajan and Al-Barzinji (2022). Thus, the polymorphism in the NR2E1/PstI gene affected egg production, body weight, and carcass weight in Iraqi local quails.

Unfortunately, there are limited studies to detect the candidate genes for economic traits of quails in Indonesia. Setiati et al. (2019) reported that the AA genotype in the growth hormone (GH/MspI) had the highest breeding value for body weight at 4 weeks of age. Rifki et al. (2021) confirmed that there were no polymorphic sites in the exon 9, iron 10, and exon 10 regions of the BMPR1B gene. An indel 24 bp mutation was detected in the promoter region of the prolactin gene and was significantly associated with the yolk color score in Indonesian quails. Furthermore, the NR2E1/PstI gene for quails in Indonesia has not been reported. This study aimed to investigate the polymorphism of the NR2E1/PstI gene in Indonesian female local quails and its association with slaughter weight, carcass weight, and carcass percentage. The results of this study are important for developing a molecular selection program in Indonesian local quails with confirmed candidate genes.

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Materials and Methods

Experimental design

Fifty female Japanese quails (Coturnix japonica) were used as experimental birds. The birds were kept under an intensive management system at PT. AqsaFarm Quail and Eel, Sentul, Babakan Madang, West Java, Indonesia. The feed nutrient standard for birds consisted of crude protein (20%–22%), crude fat (7%), crude fiber (7%), calcium (3.2%–4.0%), and phosphor (0.60%–1.0%).

DNA extraction

A total of 3 ml of blood samples were collected from each bird through the jugular vein using venoject and EDTA vacutainer tubes. The blood samples were stored in –20°C until used for the next analysis. The DNA samples of the birds were collected using a DNA extraction kit (Geneaid, Taiwan) following the manufacturer’s protocols. In the current study, approximately 1.47 of DNA purity (260/280 nm) and 2.10 ng/μl of DNA concentration were obtained according to the DNA extraction kit.

Polymerase chain reaction (PCR)

Coturnine NR2E1 gene amplification was performed in a total volume of 15 μl consisting of 4.5 μl of DNA template, 7.5 μl of DreamTaq Green PCR Mastermix (Thermo Scientific, USA), 0.75 μl of primer pairs (10 nmol), and 1.5 μl of nuclease-free water. A primer pair from Sasazaki et al. (2006) was modified to amplify the coturnine NR2E1 gene along 599 bp (Fig. 1) as follows: Forward: 5′-ACA CTA GGA ACA TAA TGG GCT-3′ and Reverse: 5′-TCA CTG TGG CGT TTC AGA TT-3′. The PCR analysis was performed in a mastercycler gradient (Eppendorf, Germany) with 1 cycle of pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, initial extension at 72°C for 30 seconds, and final extension at 72°C for 3 minutes. Subsequently, the electrophoresis analysis was assessed by 1% agarose gel staining with DNA (Florosafe DNA Stain, 1st BASE, Singapore) at 100 V for 30 minutes. DNA was visualized using the G-Box Documenation System (Syngene, UK).

Fig. 1. Primer position and PstI restriction site (CTGCA*G) along 599 bp in the target sequence of coturnine NR2E1 gene (GenBank: AB250326.1). A transversion mutation C>G was occurred in 462th nucleotide (yellow shading).

Restriction fragment length polymorphism (RFLP) and sequencing

RFLP analysis was performed in a total volume of 10 μl consisting of 2.0 μl of PCR product, 0.20 μl of PstI restriction enzyme, 1.0 μl of Tango buffer, and 6.8 μl of water free nuclease. The digestion of PstI restriction enzyme was executed at a temperature of 37°C for 1 hour with heat inactivation at 80°C for 20 minutes. For RFLP analysis, 2% agarose gel was used for electrophoresis at 80 V for 45 minutes. Thus, the G-box machine was used to observe the DNA fragments from the RFLP analysis. Sequencing analysis was performed by 1st BASE Laboratory Services (Malaysia) to confirm the mutation site in three different genotype samples.

Statistical analysis

Genetic parameters of genotype frequency, allele frequency, observed heterozygosity (Ho), expected heterozygosity (He), number of effective allele (n_e), polymorphic informative content (PIC), and chi-square (χ²) were computed in the present study, referring to Nei and Kumar (2000). The association study was analyzed using the general linear method with a mathematical formula referring to Stell and Torrie (1980) as follows:

Y_ik=μ + G_i + Ɛ_ik

where Y_ik is the observed trait, G_i is the effect of i^th genotype, and Ɛ_ik is the experimental error.

Ethical approval

The Animal Ethics Committee of the Faculty of Agriculture, Djuanda University, approved the experimental protocol (Approval Issue No: 1083/01/Faperta-A/X/2023). Ethical approval date: 23 October 2023.

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Results

Along 599 bp of the gene under study was successfully amplified on 1% agarose gel, as illustrated in Figure 2. Subsequently, sequencing analysis revealed a mutation site of g.462C>G in the PstI restriction site (CTGCA*G) of the NR2E1 gene (Fig. 3). Consequently, the RFLP analysis of the gene under study revealed three genotypes: CC (599 bp), CG (138 bp, 461 bp, 599 bp), and GG (138 bp, 461 bp), as shown in Figure 2. In general, two common alleles, C (0.42) and G (0.58), were identified in the g.462C>G mutation, with an effective n_e and a PIC of 1.95 and 0.37, respectively (Table 1). In addition, the genetic diversity in the NR2E1 gene of the studied birds was not under genetic equilibrium (χ²>5.99). Unfortunately, polymorphism in the g.462C>G of the NR2E1 gene was not significantly associated with the economic traits of the studied birds (Table 2). However, the heterozygous birds in the current study had higher carcass weight (CW) and carcass percentage (CP) traits than other genotypes. The lowest traits were observed in the CC genotype for slaughter weight and CP traits and in the GG genotype for CW traits.

Fig. 2. Amplification of Coturnine NR2E1 gene (599 bp) on 1% agarose gel (left) and PCR-RFLP results for CC gene NR2E1/PstI on 2% agarose gel (right) with three genotypes of CC (599 bp), CG (599 bp; 461 bp & 138 bp), and GG (461 bp & 138 bp). M: DNA ladder 250 bp; Line 1–4: DNA sample.

Fig. 3. Detection of g.462C>G mutation in the coturnine NR2E1 gene.

Table 1. Genetic diversity of the NR2E1/PstI gene in Japanese quails (Coturnix japonica).

Table 2. Association of NR2E1/PstI polymorphism with body and carcass weights of Japanese quails (Coturnix japonica).

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Discussion

In this study, the DNA sample with 1.47 DNA purity (260/280 nm) and 2.10 ng/μl of DNA concentration could be amplified for RFLP analysis. According to Dewanata and Mushlih (2021), the standard DNA purity and DNA concentration were 1.8–2.0 and >50 ng/µl. The purity and concentration of the DNA template can be attributed to the extraction process and DNA material sources. Previous studies have reported that the coturnine NR2E1 gene is polymorphic in Iraqi local quails (Ahmed and Al-Barzinji, 2020; Lajan and Al-Barzinji, 2022) and Japanese quails (Coturnix japonica) (Bayraktar et al., 2023). In Iraqi local quails, a primer pair from Sasazaki et al. (2006) was used to amplify the coturnine NR2E1 gene along 546 bp with the PCR-RFLP results of AA (546 bp), AC (546 bp, 404 bp, and 142 bp), and AB (546 bp, 404 bp) genotypes (Lajan and Al-Barzinji, 2022). Subsequently, a primer pair in this study was used to amplify the T-cell leukemia homeobox TLX gene in Japanese Quail, the observed genotype frequencies (AA: 0.50, AB: 0.19, BB: 0.31), this polymorphic site can be incorporated as a molecular marker into selective breeding programs for Japanese quail, with the aim of enhancing the potential for productive performance traits (Bayraktar et al., 2023).

In the present study, we confirmed that a primer pair from Sasazaki et al. (2006) was designed to detect a mutation of g.462C>G at the intronic region of Coturnine NR2E1 gene according to GenBank: AB250326.1. However, Lajan and Al-Barzinji (2022) did not show a sequence reference of Coturnine NR2E1 gene (GenBank: MN542412). Hence, the sequencing results in the intronic region of the coturnine NR2E1/TLX gene sequence from the quails under study were deposited in the GenBank database with accession ID of OR790574.1—OR790576.1. Unfortunately, studies on NR2E1/PstI gene polymorphism are limited. However, a combination of gene loci in SEMA3E, GH, and NR2E1 had a significant association with egg production, body weight, and carcass weight traits of Iraqi local quails (Ahmed and Al-Barzinji, 2020; Lajan and Al-Barzinji, 2022). Nevertheless, polymorphism of coturnine NR2E1/PstI gene in the birds under study did not influence economic traits, although this gene had high genetic diversity. A high genetic diversity can be indicated by the PIC value (Nei and Kumar, 2000).

According to the PIC value, genetic diversity can be classified in low (<0.25), moderate (0.25–0.30), and high (>0.30) categories. In addition, the n_e value reveals two common alleles (C and G) in the NR2E1/PstI gene polymorphism in the studied birds. However, the heterozygous quails had the highest CW and CP values compared with other genotypes. Despite this, the polymorphism of the NR2E1 gene also occurred in a mouse (Rattus norvegicus) and affected agressive behavior (Young et al., 2002).

When a population is in Hardy–Weinberg equilibrium, both genotype and allele frequencies remain constant from one generation to the next, assuming no evolutionary influences such as mutation, selection, or migration (Haque and Holsinger, 2013). Interestingly, the genetic diversity in the NR2E1 gene of the birds under study was not in genetic equilibrium. Genetic equilibrium can be influenced by selection, cross breeding, inbreeding, and migration (Nei and Kumar, 2000). Deviations from HWE can indicate various evolutionary forces, such as natural selection, non-random mating, genetic drift, mutation, and migration (Gupta, 2022). In general, farmers in Indonesia prefer to select day-old quail with several criteria, such as no disorder in the beak and shank, body weight of 6–8 g, normal feather shape (not dull and shiny), and not from inbreeding (Samadi et al., 2023). Consequently, the diversity of related genes, including the NR2E1 gene, was not under genetic equilibrium. Several previous studies have revealed that selection based on genetic parameters for growth and laying traits has been effective in improving these traits over generations in Ukrainian quail lines (Salih, 2024). Other studies found a negative relationship between survival and egg weight and body weight gain traits after 21 days, suggesting that improving one trait may negatively impact the other (Saghi et al., 2022). In the future, an in-depth study on the coturnine NR2E1/Pst1 gene with a large sample and record data are important to accurately obtain the genetic marker.

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Conclusion

In conclusion, the coturnine NR2E1/PstI gene was polymorphic with high genetic diversity. However, no significant association was found between NR2E1/Pst1 gene polymorphism and economic traits for Japanese quails under study. In the future, in-depth research on the coturnine NR2E1 gene involving a large sample and different gene regions is important to accurately obtain the genetic marker for economic traits.

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List of Abbreviations

CP, Carcass percentage; CW, Carcass weight; DNA, Deoxyribonucleic acid; EDTA, Ethylenediaminetetraacetid acid; GLM, General linear method; He, Heterozygosity; Ho, Heterozygosity; NR2E1, Nuclear receptor subfamily 2 group E member 1; NSCs, Neural Stem Cells system; PCR, Polymerase chain reaction; PIC, Polymorphic informative content; RFLP, Restriction fragment length polymorphism; SW, slaughter weight; TLX, T-cell leukemia homeobox; χ², Chi-square.

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Acknowledgments

The authors would like to express their sincere gratitude to the Research Center for Applied Zoology, National Research and Innovation Agency, for allowing us to conduct the research and sample analysis.

Conflict of interest

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Funding

This study did not receive funding from any source.

Authors contribution

WPBP, AB, DAL, AMD, DWS, and AR conceptualized and designed the experiment, conducted the literature review, and wrote the first draft of the manuscript. WPBP, AB, DAL, AMD, AR, DWS, IQP, MGR, and MAR edited and revised the manuscript draft. All authors have read and approved the final version of the manuscript.

Data availability

All data are provided in the manuscript.

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References

Ahmed, L.S., and Al-Barzinji, Y.M.S. 2020. Three candidate genes and its association with quantitative variation of egg production traits of local quail by using PCR-RFLP. Iraqi J. Agri. Sci. 51, 124–131.

Bayraktar, M., Baylan, M., Özcan, B.D., Kurşun, K., Dikkaya, E. and Kayaalp, G.T. 2023. Determination of TLX/PstI polymorphism in Japanese quail. CUNAS 2, 1–4.

Dewanata, P.A. and Mushlih, M. 2021. Differences in DNA purity test using UV-Vis spectrophotometer and Nanodrop spectrophotometer in type 2 diabetes mellitus patients. IJINS 15, 1–6.

Gupta, P. 2022. Population genetics. In Genetics fundamentals notes. Eds., Kar, D. and Sarkar, S. Singapore, Singapore: Springer Singapore, pp: 1077–1103.

Haque, M. and Holsinger, K.E. 2013. Equilibrium population, 2nd ed. In Brenner’s encyclopedia of genetics. Eds., Maloy, S. and Hughes, K. New York, NY: Elsevier Inc, pp: 514–6.

Ministry of Agriculture of the Republic of Indonesia. 2023. Egg Production: The number of eggs produced by poultry (free-range chickens, quails, ducks, and Manila ducks), excluding laying hens, over the course of a year, including those hatched, damaged, traded, consumed, and given to others. Available via https://satudata.pertanian.go.id/datasets

Lajan, S.A. and Al-Barzinji, Y.M.S. 2022. Detection of quantitative loci correlation with growth traits in local quail using PCR-RFLP technique. Iraqi J. Agri. Sci. 53, 16–26.

Nei, M. and Kumar, S. 2000. Molecular evolution and phylogenetics. Oxford University Press.

Rifqi, M., Cahyadi, M., Nuhriawangsa, A.M.P., Dewanti, R. and Wati, A.K. 2021. The association of BMPR1B and PRL polymorphisms with egg productivity and quality traits in Japanese quails. Poult. Sci. J. 9, 263–271.

Saghi, R., Rokouei, M., Dashab, G.R., Saghi, D.A. and Faraji-Arough, H. 2022. Using a linear-threshold model to investigate the genetic relationship between survival and productive traits in Japanese quail. Italian J. Anim. Sci. 21(1), 605–611.

Salih, J.H. 2024. Genetic parameters of growth, carcass, and laying traits in two lines of Ukrainian Qual. Iraq. J. Agri. Sci. 55(6), 1984–1993.

Samadi., Khairi, F., Ilham. and Sugito. 2023. Improving quail productivity through vaccination program and ration formulation based on local feed resources in Aceh Besar District. JPMMI 1, 32–35.

Sasazaki, S., Hinenoya, T., Lin, B., Fujiwara, A. and Mannen, H. 2006. A comparative map of macrochromosomes between chicken and Japanese quail based on orthologous genes. Anim. Genet. 37, 316–320.

Setiati, N., Mustikaningtyas, D., Dewi, N.K. and Partaya. 2019. Breeding value and GH gene frequency to four weeks old quails’ body weight. J. Phys. Conf. Ser. 1321, 32049.

Song, J., Cho, K.J., Oh, Y. and Lee, J.E. 2015. Let7a involves in neural stem cell differentiation relating with TLX level. Biochem. Biophys. Res. Commun. 462, 396–401.

Statistics of Indonesia. 2022. Peternakan Dalam Angka 2022. Jakarta, Indonesia: BPS—Statistics Indonesia.

Steel, R.G.D. and Torrie, J.H. 1980. Principles and procedures of statistics. New York, NY: McGraw-Hill.

Young, K.A., Berry, M.L., Mahaffey, C.L., Saionz, J.R., Hawes, N.L., Chang, B., Zheng, Q.Y., Smith, R.S., Bronson, R.T., Nelson, R.J. and Simpson, E.M. 2002. Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent. Behav. Brain Res. 132, 145–158.