Iranian Veterinary Journal

Iranian Veterinary Journal

Chicken Major Histocompatibility Complex Genotyping by High Resolution Melting Analysis

Document Type : Research Paper

Authors
Ammar Ramezani 1
Gholamreza Nikbakht Brujeni 2
Nariman Sheikhi 3
Kaveh Parvandar Asadollahi 3
1 PhD Student in Immunology, Department of Pathobiology, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Professor, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
3 Associate Professor, Department of Clinical Science, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran
10.22055/ivj.2026.560799.2869
Abstract
    The chicken major histocompatibility complex (MHC), known as the B-locus or serological blood group, is responsible for controlling autoimmune and resistance to viral, bacterial, and parasitic diseases. The microsatellite LEI0258, as a reliable marker for avian MHC typing, can be used to determine the identity of different populations and individual or breed characteristics in academic and applied studies. In this study, High Resolution Melting (HRM) analysis was used to determine MHC genotypes based on the LEI0258 microsatellite in Ross 308 broiler chickens. To this end, genotypes were first determined by conventional PCR and electrophoresis methods, as well as fragment analysis, and then the findings were compared and analyzed with the results of the HRM assay. From 100 samples, 5 genotypes were identified in conventional PCR and electrophoresis assays, as well as fragment analysis. These genotypes included alleles with molecular weights of 448, 385, 300, and 207 base pairs. In HRM analysis, normalized fluorescence changes versus temperature (degrees Celsius) were evaluated for five different genotypes. The results showed that the HRM method is capable of discriminating different genotypes as well as homozygous and heterozygous cases. Overall, it seems that the high-resolution melting analysis method can be applied to study the diversity of avian MHC genes. Although this technique cannot precisely identify the alleles of each chromosome, due to the co-dominant nature of MHC genes, the results are acceptable for assessing genetic composition and determining genotypes.
Keywords
Chicken
Genotyping
Major Histocompatibility Complex
High Resolution Melting Analysis
Melting Temperature Analysis

Subjects

Abdel karim, H. D., & Noori, A. A. (2025). Relationship of Lei0234 marker with some productive traits of local chickens. Iraqi Journal of Agricultural Sciences, 56(4), 1273–1285.
Alkafajy, A., Al-Karagoly, H., & Brujeni, G. N. (2020). Comparison of cattle BoLA-DRB3 typing by PCR-RFLP, direct sequencing, and high-resolution DNA melting curve analysis. Veterinary Research Forum, 11(1), 21–26.
Ben Salah, H., Jelassi, R., Zidi, I., Ben Amor, A., Bizid, S., Ammi, R., Guizani, L., Bouratbine, A., Aoun, K., & Chelbi, H. (2021). Rapid high-resolution melting method to identify human leukocyte antigen-G (HLA-G) 3′ untranslated region polymorphism +3142C/G (rs1063320). Molecular Genetics and Genomic Medicine, 9(11), e1817.
Boonyuen, U., Jacob, B. A. C., Chamchoy, K., Pengsuk, N., Talukam, S., Petcharat, C., Adams, E. R., Edwards, T., Boonnak, K., Amran, S. I., Ab Latif, N., & Louis, N. E. (2025). Improved genetic screening with zygosity detection through multiplex high-resolution melting curve analysis and biochemical characterisation for G6PD deficiency. Tropical Medicine and International Health, 30(5), 437–457.
Costamilan, C. A. da V. L. R., Paludo, E., Herkenhoff, M. E., Pértille, F., de Souza, A. L. S., Mendes, C. R., & Dilarri, G. (2025). Immunogenetic diversity of MHC class II B-Lβ genes in Brazilian Caipiras (free-range) chickens laying blue eggs. Frontiers in Immunology, 16 (10), e.1644110.
Fulton, J. E. (2020). Advances in methodologies for detecting MHC-B variability in chickens. In Poultry Science. 99(3): 1267–1274.
Fulton, J. E., Juul-Madsen, H. R., Ashwell, C. M., McCarron, A. M., Arthur, J. A., O’Sullivan, N. P., & Taylor, R. L. (2006). Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenetics, 58(5–6), 407–421.
Fulton, J. E., Lund, A. R., Mccarron, A. M., Pinegar, K. N., Korver, D. R., Classen, H. L., Aggrey, S., Utterbach, C., Anthony, N. B., & Berres, M. E. (2016). MHC variability in heritage breeds of chickens. Poultry Science, 95(2), 393–399.
Goto, R. M., Afanassieff, M., Ha, J., Iglesias, G. M., Ewald, S. J., Briles, W. E., & Miller, M. M. (2002). Single-strand conformation polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens. Poultry Science, 81(12), 1832–1841.
Manjula, P., Perera, D., Fernando, R., Kim, M., Cho, E., & Lee, J. H. (2025). Investigation of MHC-B Linked LEI0258 Marker Diversity in Various Chicken Populations. Journal of Animal Science and Technology. 10 (5), e2500118.
Mehrzad, J., & Houshmand, P. (2025). Immunogenetics Properties of Avian MHC Polymorphism and its Association with Diseases and Production Traits. Journal of Poultry Sciences and Avian Diseases, 3(3), 40–46.
Moussa Hassan, O., Machuka, E., Martina, K., Keambou Tiambo, C., Domelevo Entfellner, J.-B., & Pelle, R. (2023). Major Histocompatibility Complex Region and Diversity of the Local Chicken Populations In Niger. Journal of World’s Poultry Science, 2(4), 47–54.
Oladejo, O. A., Oseni, S. O., Kyallo, M., Entfellner, J. B. D., Tor, N. E., Tiambo, C. K., & Pelle, R. (2023). Evaluation of Genetic Variability in Four Nigerian Locally-Adapted Chicken Populations Using Major Histocompatibility Complex-Linked LEI0258 Microsatellite Marker. Poultry Science Journal, 11(2), 189–201.
Ramezani, A., Brujeni, N. G., Sheikhi, N., & Asadollahi, P. K. (2024). MHC-linked microsatellite LEI0258 variability and population structure of chicken ecotypes in Iran. Iranian Journal of Veterinary Research, 25(4), 312–318.
Słomka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G., & Strapagiel, D. (2017). High resolution melting (HRM) for high-throughput genotyping-limitations and caveats in practical case studies. International Journal of Molecular Sciences, 18(11), 2316.
Vahedi, S. M., Jamshidi, S., Lankarani Mohajer, L., & Nikbakht Brujeni, G. (2018). Allelic descrimination of canine MHC (DLA-DRB1) by high resolution melt analysis. Journal of Veterinary Research, 73(1), 93–100.
Yu, S., Cheng, Y., Tang, C. C., & Liu, Y. P. (2025). Diagnostic accuracy of high-resolution melting curve analysis for discrimination of oncology-associated EGFR mutations: a systematic review and meta-analysis. Journal of International Medical Research, 53(2), 1–15.
Zhou, Y., Ha, S., Xu, Y., Qin, X., Ma, Y., Lu, J., Wang, B., Cai, J., Duan, Z., Cong, B., Chen, J., & Deng, J. (2025). Establishment of a simple prediction method for DNA melting temperature: high-resolution melting curve analysis of PCR products. PLoS One, 20(4):e0321885.