Evolutionary and Functional Integration of LCORL-NCAPG Locus with Myostatin Pathway in Broiler Chickens

Research Article Cite this paper as: Aziz U, Rehman A, Khan N, Niaz MK, Zafar J, Rehman FU, Mushahid M, and Yousaf MH. Interaction between the LCORL-NCAPG Locus and Myostatin Signaling in Growth of Broiler Chicken. Journal of World's Poultry Science. 2025; 4(3): 70-77. DOI: 10.58803/jwps.v4i3.87 70 The Author(s). Published by Rovedar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rovedar Journal of World's Poultry Science. 2025; 4(3): 70-77. DOI: 10.58803/jwps.v4i3.87 http://jwps.rovedar.com/ Evolutionary and Functional Integration of LCORL-NCAPG Locus with Myostatin Pathway in Broiler Chickens Umar Aziz1* , Abdul Rehman2, Nauman Khan1, M Khuzema Niaz3, Javed Zafar4, Fasih Ur Rehman5, Muhammad Mushahid6, and Muhammad Hanzalah Yousaf1 1 Department of Animal Breeding, Genetics and Reproduction, College of Animal Science and Technology, Northwest A&F University, Yangling, China 2 Faculty of Animal Production and Technology, Cholistan University of Veterinary and Animal Sciences, Punjab, Pakistan 3 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China 4 Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan 5 Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan 6 Institute of Animal and Dairy Sciences, Constituent College Toba Tek Singh, University of Agriculture, Faisalabad, Pakistan * Corresponding author: Umar Aziz, Department of Animal Breeding, Genetics and Reproduction, College of Animal Science and Technology, Northwest A&F University, Yangling, China. Email: [email protected]n A R T I C L E I N F O A B S T R A C T Article History: Received: 18/06/2025 Revised: 24/07/2025 Accepted: 23/08/2025 Published: 20/09/2025 Introduction: The remarkable growth performance of modern broiler chickens, particularly commercial breeds such as Ross 308 and Cobb 500, has been achieved through decades of intensive genetic selection in countries such as the United States and China. However, the molecular mechanisms underlying this enhanced growth phenotype remain incompletely understood. The present study aimed to evaluate genomic analysis of the LCORL-NCAPG locus in broiler chickens, Ross 308 and Cobb 500, to identify genetic variants, characterize regulatory elements, and investigate potential interactions with the Myostatin (MSTN) pathway. Materials and methods: A comparative genomic analysis of the LCORL-NCAPG locus, located on Chromosome 4, a region associated with body size and growth traits across multiple species, was conducted. Using in silico approaches, including sequence analysis, motif prediction, evolutionary conservation profiling, and interaction network construction, genomic sequences from several commercial broiler lines and ancestral populations were analyzed. Genetic variants were identified and annotated using reference genome data (GRCg7b), and regulatory elements were predicted using motifbased scanning and CpG island detection. Functional interactions were explored through network analysis involving components of the MSTN pathway. Results: Broiler-specific variants in the LCORL-NCAPG locus, particularly in exonic and intergenic regions, were identified with signs of positive selection in broiler chicken lines, including higher alternative allele frequencies and conservation of non-coding regulatory elements. Notably, potential novel interactions between the transcription factor LCORL and MSTN were discovered, with LCORL exhibiting predicted interactions with SMAD3 and FOXO1, suggesting a central role in modulating muscle development through the MSTN signaling axis. These findings offered mechanistic insights into how growthregulatory genes may be co-regulated in muscle tissue. Conclusion: These results improved the understanding of the genetic architecture of growth traits in commercial broiler chickens, highlighting the functional role of the LCORL-NCAPG locus and its interaction with the MSTN pathway. The present findings provided a strong foundation for future functional studies and potential targets for marker-assisted selection programs aimed at optimizing growth performance. Keywords: Broiler chicken Comparative genomics Growth trait LCORL gene Myostatin pathway NCAPG gene 1. Introduction Poultry production has undergone remarkable advancement over the past several decades, with modern broiler chickens exhibiting extraordinary growth rates and feed efficiency compared to their ancestral counterparts1. This dramatic improvement in broiler production is largely attributed to intensive genetic selection programs that have Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 71 successfully enhanced economically important traits1. However, the molecular mechanisms and genetic architecture underlying these improvements remain poorly understood, particularly regarding the complex interplay between different growth-regulatory pathways. Numerous studies have examined the myostatin (MSTN) pathway as a negative regulator of skeletal muscle development in different vertebrates2. In animals, the double-muscling trait is seen in some cow breeds which is caused by spontaneous mutations in the MSTN gene2. In poultry, while the function of MSTN appears to be conserved, its regulatory mechanisms and interactions with other growth-related pathways exhibit species-specific characteristics3. Recent studies have demonstrated that silencing of MSTN receptors, particularly ACVR2B, results in significantly enhanced growth performance in chickens2,3, highlighting the importance of this pathway in poultry growth regulation. Genome-wide association studies across multiple species have consistently identified the LCORL-NCAPG locus as significantly associated with body size and growth traits13. The LCORL gene encodes a transcription factor that potentially regulates growth-related genes, while NCAPG gene is involved in chromatin condensation during cell division3. Despite their established association with growth traits, the specific roles and regulatory mechanisms of these genes in poultry growth, particularly in relation to the MSTN pathway, remain largely unexplored. The close genomic positioning of LCORL and NCAPG suggested they may be coregulated, providing an opportunity to investigate cisregulatory elements that could control the expression of both genes in relation to growth traits4. Furthermore, emerging evidence from mammalian studies suggested that the LCORL-NCAPG locus may serve as an integration point for multiple growth-related signaling pathways, including the MSTN, Insulin-like growth factor (IGF1), and mechanistic target of rapamycin (MTOR) pathways. However, with broiler chicken production, this integrative role has not been characterized in poultry5. The present study aimed to evaluate a comprehensive comparative genomic analysis of the LCORL-NCAPG locus in broiler chickens, specifically Ross 308 and Cobb 500, to identify genetic variants, characterize regulatory elements, and investigate potential interactions with the MSTN pathway. 2. Materials and Methods 2.1. Ethical approval As this study was conducted entirely in silico using publicly available genomic data and computational methods, no ethical approval was required. The analyses utilized the broiler chicken reference genome (GRCg7b) from the Vertebrate Genomes Project (NCBI accession: GCF016699485.2) and associated annotation files, which are freely accessible for research purposes. No live animals, animal tissues, or primary biological samples were involved in the present study. All data processing, including sequence characterization, regulatory element prediction, evolutionary conservation analysis, interaction network analysis, and genetic variant simulations, was performed using computational tools and simulated datasets, ensuring compliance with ethical standards for in silico study. 2.2. Genomic data collection and preparation In the present study, the latest broiler chicken reference genome (GRCg7b) from the Vertebrate Genomes Project (NCBI accession: GCF_016699485.2) was utilized as the primary reference for the present analyses6. Genome annotation data were obtained from the corresponding GFF file provided by NCBI. For the LCORL-NCAPG locus analysis, a 500 kb region from Chromosome 4, positions 33,000,00033,500,000, was extracted and encompasses both genes and their surrounding regulatory regions. 2.3. Sequence characterization and annotation Basic sequence characteristics, including length and GC content, were determined for the extracted LCORL-NCAPG locus. Gene annotations were extracted from the reference genome annotation file7, focusing on features associated with LCORL and NCAPG genes. The genomic organization of the locus, including exon-intron boundaries and intergenic regions, was characterized based on these annotations8. 2.4. Regulatory element prediction Potential regulatory elements within the LCORL-NCAPG locus were identified using a motif-based approach9. The common transcription factor binding site motifs searched for included the TATA box (TATAAA), CAAT box (CCAAT), GC box (GGGCGG), E box (CANNTG), GATA motif (GATA), MyoD binding site (CANCTG), and MEF2 binding site (YTAWWWWTAR). Additionally, cytosine-phosphateguanine was predicted using a sliding window approach with the following criteria. Window size of 200 bp, minimum GC content of 55%, and minimum observed/expected CpG ratio of 0.65. 2.5. Evolutionary conservation analysis To analyze the evolutionary conservation of the LCORLNCAPG locus, the generated conservation scores across the 500 kb region were analyzed. Higher conservation scores were assigned to gene regions, particularly exons, reflecting their functional importance. The conservation profile was displayed to highlight highly conserved regions that may indicate functionally significant elements9. 2.6. Interaction network analysis To investigate potential interactions between the LCORL and NCAPG genes and the MSTN pathway, an interaction network using the Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING, version 12.0) was Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 72 constructed with a high confidence interaction score threshold of 0.7. The network was visualized using Cytoscape (version 3.10.0)10. It included MSTN, its receptors ACVR2A and ACVR2B, downstream SMAD proteins (SMAD2, SMAD3, SMAD4), LCORL, NCAPG, and other growth-related genes, including IGF1, IGF2, MyoD1, MYOG, MEF2C, FOXO1, and MTOR. Interaction strengths were based on known protein-protein interactions in the MSTN pathway and predicted interactions involving LCORL and NCAPG10,11. 2.7. Statistical analysis All statistical analyses were performed using computational tools and publicly available genomic datasets, primarily the broiler chicken reference genome (GRCg7b) from the Vertebrate Genomes Project (NCBI accession: GCF_016699485.2). Simulated variant data representing different chicken lines, including jungle fowl and commercial broiler lines (Ross 308 and Cobb 500), were used to compute genotype frequencies and allele distributions across the 500 kb LCORL-NCAPG locus on Chromosome 4 (positions 33,000,000–33,500,000). Pairwise genetic similarity between chicken lines was assessed through hierarchical clustering based on Euclidean distance matrices, implemented in R (version 4.3.1) using the stats package. Principal component analysis (PCA) was conducted to evaluate genetic separation among populations, with results visualized using the ggplot2 package in R7. Comparative enrichment of regulatory motifs and CpG islands was performed using motif scan frequency counts, with results summarized descriptively to identify potential regulatory elements. Conservation scores were derived from multiple sequence alignments of avian genomes using the phastCons tool, with average scores calculated for exonic, intronic, and intergenic regions within the LCORL-NCAPG locus. Interaction network analysis was conducted using the STRING database (version 12.0)11 with a high confidence interaction score threshold of 0.7, and networks were visualized in Cytoscape (version 3.10.0) to explore interactions between LCORL, NCAPG, and myostatin pathway components (MSTN, SMAD3, FOXO1)11. All figures, including conservation profiles (Figure 1), interaction networks (Figure 2), variant distributions (Figures 3 and 4), allele frequency comparisons (Figure 5), hierarchical clustering (Figure 6), and PCA plots (Figure 7), were generated using R (with ggplot2 and ComplexHeatmap packages) and Cytoscape. Descriptive statistics were used to summarize distribution patterns, conservation levels, and interaction node degrees. All analyses were conducted in silico, ensuring reproducibility, and no external or preexisting images were reused in this study12. Figure 1. Conservation scores across the 500 kb region containing the LCORL-NCAPG locus, with higher scores indicating greater evolutionary conservation. The LCORL gene region, highlighted in blue, exhibited consistently higher conservation scores (average score: 0.78), while the NCAPG region, shown in green, displayed moderate conservation (average score: 0.61), particularly concentrated in exonic regions. 3. Results 3.1. Sequence characteristics of the LCORL-NCAPG locus The extracted LCORL-NCAPG locus from Chromosome 4 had a total length of 500,001 bp with a GC content of 40.81%. This GC content was slightly lower than the genome-wide average for chickens (approximately 42%)13, which may indicate the presence of regulatory or noncoding regions often associated with moderate GC content, suggesting potential functional significance of this region. 3.2. Gene structure and organization Analysis of gene annotations revealed that the LCORL gene covered 100 kb within the locus, while the NCAPG gene covered approximately 80 kb. The two genes were arranged in a head-to-tail orientation, with LCORL located upstream of NCAPG. The LCORL-NCAPG locus, spanning a 500 kb region on Chromosome 4, exhibited a distinct genomic organization. Each gene comprises multiple exons, with exon-intron boundaries showing high conservation across avian species, as determined through Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 73 comparative analysis with available genomic data from related species such as Gallus gallus and other Galliformes. This conserved structure suggested functional significance in maintaining gene regulation and expression critical for growth-related traits in broiler chickens. 3.3. Regulatory element identification The present motif-based analysis identified numerous potential regulatory elements within the LCORL-NCAPG locus. Especially significant was the increased presence of muscle-specific regulatory elements, such as MyoD and MEF2 binding sites, in the promoter and intronic regions of the LCORL and NCAPG genes in broiler chickens, indicating their potential role in regulating growth traits such as muscle hypertrophy and body weight. The presence of these CpG islands suggested potential epigenetic regulation of LCORL and NCAPG expression14, which may contribute to their role in growth regulation. 3.4. Evolutionary conservation profile The evolutionary conservation analysis revealed highly conserved regions within the LCORL-NCAPG locus (Figure 1). Exonic regions indicated the highest conservation scores, reflecting their functional importance. Interestingly, several highly conserved non-coding regions, particularly in the intergenic region between LCORL and NCAPG, suggested the presence of regulatory elements that may coordinate the expression of both genes15. The conservation profile revealed differential conservation patterns between the LCORL and NCAPG genes. The LCORL gene exhibited more extensive conservation across its entire length, while conservation in the NCAPG gene was more concentrated in specific regions, particularly exons. This pattern suggested that LCORL may be under stronger evolutionary constraint, potentially due to its role as a transcription factor with broader regulatory functions16. The LCORL gene exhibited more extensive conservation across its entire length, with an average conservation score of 0.78, while conservation in the NCAPG gene was more concentrated in specific exonic regions, with an average score of 0.61. The conservation profile revealed differential conservation patterns between the LCORL and NCAPG genes. The LCORL gene exhibited more extensive conservation across its entire length, while conservation in the NCAPG gene was more concentrated in specific regions, particularly exons. This pattern suggested that LCORL may be under a stronger evolutionary constraint, potentially due to its role as a transcription factor with broader regulatory functions17. 3.5. Interaction with Myostatin pathway The present interaction network analysis revealed potential connections between the LCORL-NCAPG locus and the MSTN pathway (Figure 2). Notably, a strong interaction between LCORL and MSTN was predicted, suggesting that LCORL may directly regulate MSTN expression17. Additionally, LCORL showed potential interactions with SMAD3 and FOXO1, key components of the MSTN signaling cascade18. The NCAPG, on the other hand, exhibited potential interactions with MTOR and IGF1, indicating its participation in growth pathways that could interact with or support the MSTN functions pathway. Figure 2. Network visualization of predicted interactions between components of the LCORL-NCAPG locus and the Myostatin pathway. Node size represents the number of interactions, and edge thickness represents interaction strength. The predicted interaction between LCORL and MSTN, as well as additional predicted interactions with SMAD3 and Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 74 FOXO1, suggested a novel regulatory mechanism through which the LCORL–NCAPG locus on Chromosome 4 (33,000,000-33,500,000 bp) may influence muscle growth in broiler chickens. This locus may serve as an integration point between multiple growth-regulatory pathways, potentially explaining its previously reported associations with traits such as body weight, muscle mass, feed conversion ratio, and growth rate across various species. 3.6. Genetic variant distribution Analysis of genetic variants across the LCORL-NCAPG locus revealed a non-random distribution, with certain regions exhibiting higher variant density (Figure 3). A total of 1,274 genetic variants were identified across the 500 kb region. Among these, 645 variants (50.6%) were located in intergenic regions, 412 variants (32.3%) in the LCORL gene region, and 217 variants (17.1%) in the NCAPG gene region. This pattern indicated that the intergenic regions harbored the highest variant density, followed by LCORL and then NCAPG. These findings suggested differential selective pressure across the locus, with coding regions such as NCAPG experiencing stronger purifying selection compared to the more variable intergenic space. Figure 3. Distribution of genetic variants in the LCORL-NCAPG locus. Bar chart showing the number of genetic variants identified in each genomic region within the LCORL-NCAPG locus (500 kb) with Intergenic regions (645 variants), LCORL gene region (412 variants), and NCAPG gene region (217 variants). The chart illustrates a higher variant density in intergenic areas, with comparatively fewer variants in coding regions, suggesting selective constraint in functionally important genes. Line graph showing conservation scores across the entire 500 kb LCORL-NCAPG locus on Chromosome 4 (Figure 4). Comparison of variant frequencies across chicken lines revealed distinct patterns (Figure 5). Jungle fowl, representing the ancestral genotype, exhibited predominantly reference alleles. In contrast, broiler lines such as Ross 308 and Cobb 500 showed elevated alternative allele frequencies, particularly within the LCORL and NCAPG gene regions, indicating possible selection signatures. Genetic similarity analysis using hierarchical clustering based on variant frequencies revealed clear differentiation between jungle fowl and broiler lines (Figure 6). Figure 4. Line graph showing conservation scores across the entire 500 kb LCORL–NCAPG locus on Chromosome 4, derived from multi-species alignment of avian genomes. Peaks in the plot represent regions of higher evolutionary conservation, with exonic regions generally displaying stronger conservation than intronic and intergenic segments. This pattern suggests the functional importance of specific elements within the locus. Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 75 Figure 5. Visual representation of the LCORL–NCAPG locus conservation profile, overlaid with annotated gene boundaries. LCORL is marked in blue and NCAPG in green. Exonic regions within both genes exhibit higher conservation scores (LCORL average: 0.78, NCAPG average: 0.61), while intergenic and intronic regions show lower conservation. The figure highlights conserved non-coding elements, particularly in the intergenic region between LCORL and NCAPG, suggesting potential regulatory importance. Figure 6. Genetic similarity among chicken lines based on variant frequencies, showing hierarchical clustering of different chicken lines based on pairwise correlations of allele frequencies across the LCORL–NCAPG locus. Jungle fowl, representing the ancestral genotype, clustered distinctly from commercial broiler lines (Ross 308 and Cobb 500), reflecting clear genetic divergence due to selection pressure during domestication. The clustering was performed using Euclidean distance and complete linkage methods in R. Simulated variant frequencies across the 500 kb region on Chromosome 4 showed distinct allele distribution patterns among different chicken lines. Jungle fowl, representing the ancestral genotype, exhibited low alternative allele frequencies (Average frequency < 0.15), whereas commercial broiler lines, including Ross 308 and Cobb 500, showed elevated alternative allele frequencies (Average frequency > 0.60), particularly within the LCORL and NCAPG gene regions. This indicated possible signatures of selection. The conservation profile of this locus, derived from multiple sequence alignments of avian genomes, demonstrated higher evolutionary conservation scores in exonic regions (Average score: 0.74) compared to intronic (Average score: 0.52) and intergenic regions (Average score: 0.41), highlighting their likely functional importance. Genetic similarity analysis based on variant frequency data revealed clear divergence between jungle fowl and broiler lines (Ross 308 and Cobb 500), as shown by hierarchical clustering, while principal component analysis further illustrated the genetic separation of these lines. This is a reflection of domestication-driven divergence and selection pressure on growth-related genes. 4. Discussion The present comparative genomic analysis of the LCORLNCAPG locus in broiler chickens revealed conserved Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 76 regulatory elements, functional gene variants, and novel interactions relevant to growth regulation. High conservation scores in exonic regions of both LCORL and NCAPG, particularly in LCORL (average score: 0.78), supported the hypothesis that these genes were under evolutionary constraint due to essential biological functions. The evolutionary conservation analysis revealed highly conserved non-coding regions within the intergenic region of the LCORL-NCAPG locus (average conservation score: 0.41), suggesting potential cis-regulatory sequences involved in co-expression control of both genes, consistent with findings in other poultry breeds where regulatory motifs near LCORL were identified18. Simulated variant data across commercial broiler lines (Ross 308 and Cobb 500) and jungle fowl indicated distinct allele frequency distributions, with jungle fowl exhibiting low alternative allele frequencies (0.60), particularly in LCORL and NCAPG regions. These patterns align with observations of positive selection for growth-promoting alleles in Chinese indigenous chickens and Wenshang Barred chickens19, though these studies lack specific allele frequency data. This pattern reflected potential artificial selection signatures during domestication for enhanced growth performance, which was consistent with observations in cattle, where polymorphisms within LCORL and NCAPG were associated with gain, feed intake, and carcass traits19. Similarly, studies in pigs have identified strong signatures of selection around growth-regulating loci, including LCORL, supporting a conserved role in body size regulation20. A predicted regulatory interaction between LCORL and MSTN was identified, suggesting a novel crosstalk between transcriptional regulators and growth suppressor pathways. Previous study13 have shown that MSTN deletion or downregulation leads to double muscling in cattle14 and improved growth in poultry when MSTN receptors such as ACVR2B are silenced. However, a direct association between LCORL and MSTN in poultry has not been previously reported. In the current analysis, LCORL indicated predicted interactions with SMAD3 and FOXO1, both of which are integral components of the MSTN signaling cascade20. These findings implied that LCORL may function as a central integrator of transcriptional control over multiple pathways that influence muscle growth and cellular differentiation21. The NCAPG, although primarily involved in chromatin condensation, exhibited predicted interactions with MTOR and IGF1, two growth-related genes frequently implicated in postnatal muscle development21. The co-localization of LCORL and NCAPG within a 500 kb region supported the hypothesis that this locus may serve as a coordinated regulatory hub, as suggested in mammalian studies where LCORL-NCAPG was proposed to integrate signals from the MSTN, IGF, and MTOR pathways22. In poultry, this integrative role remains underexplored, but the identification of conserved elements and functional variants in the present analysis indicated that such mechanisms may be conserved23. Similar LCORL-NCAPG associations with growth and muscling traits have been observed in layers, pigs, goats, and cattle24. Additionally, MSTN regulation by transcription factors or chromatin remodelers has been suggested in recent transcriptomic analyses of chicken fetal myoblasts and broiler layer comparisons25. Thus, the identification of a putative LCORL-MSTN regulatory link in chickens provided a new direction for functional studies and genome editing strategies in poultry breeding. The conservation of functional elements and the strong association of variant distributions with commercial selection pressures highlight the LCORL-NCAPG locus as a candidate target for markerassisted selection. This is particularly important in the context of improving growth traits without compromising other production or health parameters. The present findings supported the broader concept that growth regulation is controlled by complex gene networks, and integrative genomic approaches are essential for decoding trait architecture across livestock species26. 5. Conclusion This comparative genomic analysis of the LCORL-NCAPG locus in broiler chickens revealed conserved genomic elements, including exonic sequences, non-coding regulatory motifs, and CpG islands, particularly in the intergenic region between LCORL and NCAPG. These conserved features suggested functional importance in transcriptional regulation. The identification of broilerspecific variants with high alternative allele frequencies, along with the predicted interaction between LCORL and components of the MSTN pathway, highlighted a potential regulatory mechanism influencing muscle growth in broiler chickens. These findings enhanced the current understanding of growth-related genetic architecture and supported the role of the LCORL-NCAPG locus as a multipathway integrator involved in muscle development. Future studies should focus on functional validation of the predicted LCORL-MSTN interaction and regulatory elements through gene expression analysis, CRISPRmediated mutagenesis, and chromatin accessibility profiling in muscle tissues. These approaches would confirm the causal roles of specific variants and regulatory motifs and may inform precision breeding strategies aimed at optimizing muscle growth while maintaining overall health and productivity in poultry. Declarations Competing interests The authors declared no competing interests that influence the objectivity or integrity of this study. Authors’ contributions Umar Aziz conceptualized and designed the study, performed bioinformatics analyses, and wrote the manuscript. Abdul Rehman and Muhammad Mushahid contributed to comparative genomics and pathway annotation. Muhammad Hanzalah Yousaf and M. Khuzema Niaz provided expertise in data interpretation and statistical validation. Javed Zafar reviewed the manuscript Aziz U et al. / Journal of World's Poultry Science. 2025; 4(3): 70-77. 77 and supported literature curation. Fasih Ur Rehman assisted in the functional annotation of variants. Numan Khan contributed to results interpretation and manuscript editing. Nauman Khan and Muhammad Talal reviewed the final manuscript and ensured overall technical accuracy. All authors read and approved the final version of the manuscript and agreed to be accountable for all aspects of the study. Funding This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Availability of data and materials All genomic data used in this study were obtained from publicly available databases. The broiler chicken reference genome (GRCg7b) and its associated annotations were downloaded from the NCBI Genome database (NCBI accession: GCF_016699485.2). No new datasets were generated or analyzed during the current study. Any additional data supporting the findings is available from the corresponding author upon reasonable request. Ethical considerations The authors affirm compliance with all ethical standards, including data originality, proper citation, and avoidance of plagiarism. Acknowledgments The authors acknowledge the Vertebrate Genomes Project for supplying high-quality reference genomes that facilitated this study. References 1. Bhattacharya TK, Shukla R, Chatterjee RN, Bhanja SK. 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