eng Rovedar Journal of World’s Poultry Science 2980-7999 2025-09-01 4 3 30 42 10.58803/jwps.v4i3.79 81 Umar Aziz 0 https://orcid.org/0009-0009-6266-4340 Abdul Rehman 1 https://orcid.org/0009-0009-3221-2984 Muhammad Mushahid 2 https://orcid.org/0000-0001-8670-3011 Fasih Ur Rehman 3 https://orcid.org/0000-0002-7750-3453 Nauman Khan 0 https://orcid.org/0009-0008-1289-6859 Muhammad Hanzalah Yousaf 4 https://orcid.org/0009-0005-7241-5701 M Khuzema Niaz 5 https://orcid.org/0009-0009-2039-3570 Muhammad Arslan Akbar 0 https://orcid.org/0000-0001-5418-1271 Muhammad Rizwan 0 https://orcid.org/0009-0004-9832-9193 Saleh Ahmad 6 https://orcid.org/0009-0007-0595-4320 Department of Animal Breeding, Genetics and Reproduction, College of Animal Science and Technology, Northwest A&F University, Yangling District, Xianyang, Shaanxi, China Department of Animal production and technology, Cholistan University of Veterinary and Animal Sciences - CUVAS Bahawalpur, Pakistan Department of Animal Breeding and Genetics, University of Agriculture Faisalabad, Constituent College Toba Tek Singh, Pakistan Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan Faculty of Animal Husbandry, University of Agriculture, Faisalabad, Pakistan Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China Department of Zoology, University of Education, Faisalabad Campus, Pakistan Introduction: Poultry layer breeds have undergone extensive selection for egg production traits, yet the genetic basis of many economically important characteristics remains incompletely understood. In the present study, a comprehensive comparative genomic analysis of multiple commercial and indigenous poultry layer breeds was conducted to identify novel genes associated with egg production and disease resistance, such as viral and bacterial infections, particularly through innate immune pathways. Materials and methods: A comprehensive comparative genomic analysis was conducted using whole-genome sequencing data from 135 individuals, including 30 commercial layers (White leghorn and Rhode Island red), 90 indigenous chickens from six local breeds, and 15 red jungle fowl representing the ancestral population. Using fixation index scans, haplotype-based selection analysis, and Tajima’s D, genomic regions under positive selection were identified. By integrating gene prediction tools and protein function analysis, 12 novel genes in layer breeds with strong selection signals and potential roles in egg production and immune response were identified. Results: In silico functional analysis, utilizing protein domain annotation, structural modeling, and pathway enrichment, suggested that these novel genes are involved in eggshell formation, egg production, immune response, and metabolic regulation. Notably, GALLUS-NOVEL-3, a previously uncharacterized gene with high predicted oviduct expression and strong selection signatures in commercial layers, may play a role in calcium transport and eggshell mineralization. Protein structure prediction and domain analysis further supported the potential functionality of these novel genes, revealing conserved features linked to reproductive physiology and immune defense.        Conclusion: The present findings provided new insight into the genetic basis of economically important traits in layer chickens and highlight promising targets for breeding programs aimed at improving egg production and disease resistance. https://jwps.rovedar.com/index.php/JWPS/article/view/79 Comparative genomic Disease resistance Egg production Novel gene Poultry layer Selection signature