Libyan local chickens are an integral part of the agricultural heritage of Libya. These indigenous chickens are recognized for their ability to survive in harsh environmental conditions. Therefore, these local chickens are very important for the rural population of Libya (Manyelo et al., 2020). Generally, local chickens are divided based on the phenotypic characteristics of the birds, such as the size of the birds, feathers, comb type, color of the shank, and plumage color. However, there are no genetic classifications of local chickens (Ariza et al., 2021). Only a few local chickens have been classified as ecospecies based on the phenotypic characteristics of the birds (Chebo et al., 2024). Therefore, the genetic background of local chickens needs to be considered for the purpose of breeding, conservation, and utilization.

This technique has been found to be an efficient tool for understanding the genetic structure of an organism. It is also used to study the genetic diversity, evolutionary relationships, and genes associated with traits (Jones & Wilson, 2022); (Xu et al., 2023). Recently, the advancement of Pylogenetic Analysis sequencing has helped to improve the efficiency of the WGS technique. This technique is able to detect complex structural variations of the genome (Wang et al., 2021). It has been found to be a faster, cost-effective, and accurate method compared to the other two sequencing technologies, i.e., PacBio and Illumina (Satam et al., 2023). The analysis of mitochondrial DNA (mtDNA) has also been found to be an efficient tool for understanding the genetic diversity of an organism (Myćka et al., 2022). Indigenous cattle have been found to have desirable traits such as resistance to diseases and adaptability to the climate. The genetic diversity of indigenous cattle is under threat due to the presence of commercial breeds (Taye, 2024). Hence, the genomic study of the cattle species is required (Bist et al., 2024); (Allayee et al., 2023); (Yevshin et al., 2023). This research aims to contribute to the preservation of the Libyan chicken as a valuable genetic resource and support the development of more sustainable and resilient poultry farming systems.

Describing Libyan chicken gene content, organization, and nucleotide pattern was accomplished through successful interrogation of its entire mitochondrial DNA (mtDNA) genome sequence. The mitogenome includes ribosomal RNA (rRNA) genes, transfer RNA (tRNA) genes, and protein-coding genes. These elements are needed for mitochondrial operation, including oxidative phosphorylation and energy metabolism. Table 1 shows the whole sequence profile, including nucleotide composition, strand orientation, codon use, and gene length. Comparative genomic analyses with other species of chicken are made easy by this information, which also gives vital information about the molecular profile of the Libyan chicken. The mitochondrial genome of the Libyan chickens was assembled with a total length of 17,017 bp, contrast to the chicken mitochondrial genomes used for comparison (16,775–16,788 bp), This difference in the size of the mitochondrial genome can mainly be attributed to variations in the length of the non-coding control region (D-loop), because this region has been shown to have insertions and deletions and tandem repeats among different populations of chickens.

As shown in Table 1, the Libyan chicken mitochondrial genome has the usual protein-coding genes (COX1, COX2, COX3, ATP6, ATP8, ND1, ND2, ND3, ND4, ND4L, ND5, and CYTB). The gene ND5 is the largest, with 1814 bases, while the gene ATP8 is the smallest, with 165 bases. Most of the genes start with the codon ATG, but ATT and ATC are also present, and the stop codon TAA predominates. Most of the genes are located on the heavy strand, but the gene ND6 is located on the light strand. The nucleotide composition is somewhat biased, with the gene ND6 having unusually high levels of thymine and guanine. Statistical tests confirmed the significant difference in the composition of the nucleotides among the genes and the specific composition of the gene ND6 compared to the protein-coding genes.

Comparison of the mitochondrial genome of Libyan chickens with those of 27 breeds and their wild ancestor, Gallus gallus bankiva, is shown in Table 2. The Libyan chicken’s mitogenome is longer, with 17,017 base pairs, compared to the other breeds, which have 16,775 to 16,788 base pairs. However, the Libyan chicken’s nucleotide composition is similar to that of the other breeds, with C, A, T, and G at 32.5%, 30.2%, 23.7%, and 13.5%, respectively. Statistical analysis showed that gene identity has a significant impact on the composition of the nucleotides, with the Libyan chicken’s ND6 gene having a distinct composition with higher levels of thymine and guanine. The Libyan chicken’s nucleotide composition is similar to some Chinese local breeds and the Red Jungle Fowl, but has a longer mitogenome, which could be a distinct genomic feature for the study of genetic identity.

The mitochondrial genome reference sequences used to compare the Libyan chickens with other breeds are shown in Table 3. The Libyan chicken genome sequence was characterized in the present study, while the other sequences were obtained from the GenBank database and the literature. These reference sequences include Asian native breeds, Indian indigenous breeds such as Aseel, Tellichery, Nicobari, Haringhata Black, and Ghagus, commercial breeds such as Silky, White Leghorn, and Brahma, and wild ancestors such as Gallus bankiva and Red Jungle Fowl. The references include studies from 2002 to 2023, and the references were obtained from various research groups. Some of the studies were conducted to compare the domestication and phylogenetic relationships, such as (Miao et al., 2013) and(Liu & Zhao, 2023).

Figure 2 shows a circular map of the Libyan chicken mitochondrial genome sequence obtained using the Pylogenetic Analysis. The outer circle represents various gene annotations, including protein-coding genes, tRNA, and rRNA. The second circle represents nucleotide sequence, with inner circles showing GC content and AT content distribution, respectively, which are essential in understanding genomic variations and stability. Although mitochondrial genome structure is conserved in chickens, genetic variations are evident (Gao et al., 2025). This genomic sequence shows that Libyan chickens are evolutionarily unique, thus essential for biodiversity and breeding purposes (Moftah et al., 2025).

As presented in Figure 3, the genetic distance matrix for the pairwise genetic distances between Libyan chickens and the other local and commercial breeds is depicted. The lower the values, the closer the genetic relationship between the breeds, whereas the higher the values, the more genetically distant the breeds are from each other. The values revealed that the Libyan chickens have high genetic distances from the majority of the local and commercial breeds, including the White Plymouth Rock, White Layer, and Broilers, implying low genetic admixture. However, moderate genetic distances from some indigenous breeds such as Kadaknath, Kedu, and Aseel suggest a possible genetic connection. The genetic distance matrix verifies the uniqueness of the Libyan chickens as a genetically distinct breed with high conservation value.

Figure 4 shows a phylogenetic tree constructed from D-loop sequence analysis for the evolutionary relationships of Libyan chickens compared to different native and commercial breeds. Libyan chickens have a unique genetic makeup but are closely related to some of the indigenous Asian chicken breeds, such as Gushi, Chigulu, Rugao Yellow, etc. They are also closely related to some of the wild chicken species, such as the Red Jungle Fowl and Gallus Bankiva. The Libyan chickens are not closely related to the commercial breeds such as White Leghorn and White Plymouth Rock. Figure 4 represents the phylogenetic tree generated through the analysis of the 801 bp-long sequence of the mitochondrial D-loop of the Libyan chicken, along with 27 other domesticated, indigenous, and commercial chicken breeds. Phylogenetic analysis was performed on D-loop sequences aligned using the maximum-likelihood approach. The Libyan chicken appeared as a distinct lineage, although it was associated with several indigenous Asian chicken breeds, such as Gushi, Chigulu, and Rugao Yellow. The genetic distance of the Libyan chicken was found to be closer to that of its wild ancestors, such as the Red Jungle Fowl and Gallus bankiva. However, the genetic divergence of the Libyan chicken was greater from the commercial chicken breeds such as White Leghorn and White Plymouth Rock.

Figure 5 is a phylogenetic tree based on whole-genome sequence data showing the evolutionary relationships between Libyan chickens and other chicken breeds. As seen in the figure, Libyan chickens have their own evolutionary branch, showing a high genetic divergence rate compared to both domestic and indigenous chicken breeds. Unlike the Nicobari, Ghagus, and Aseel breeds, which are closely related, Libyan chickens are isolated in terms of their genetic information. This means that geographical isolation and adaptation to the environment, as well as low rates of crossbreeding, have influenced the formation of the chicken genome. Figure 5 illustrates the maximum-likelihood phylogenetic tree developed from the complete sequence of the mitochondrial genome (mtDNA) (17,017 bp) of the Libyan chicken and the complete mitochondrial genome sequence of 27 chicken reference breeds available in GenBank. The study was based solely on the mitochondrial genome, excluding any nuclear genomic sequences. From the phylogenetic tree, the Libyan chicken appears to have evolved into a separate lineage, showing significant genetic divergence from other indigenous and commercial chicken breeds. Although the Nicobari, Ghagus, and Aseel chickens were grouped into a single cluster, the Libyan chicken formed a separate cluster, indicating a distinct maternal evolutionary lineage.

The present study is the first comprehensive analysis of the whole-genome sequence of the Libyan chicken using phylogenetic analysis. The findings bring significant new knowledge about the genetic composition, variability, and evolutionary position of this indigenous breed. With the aid of the MEGA tool for evolutionary genetic analysis augmented by genome assembly pipelines such as Minimap2, SAMtools, Prokka, and BCFtools, the genomic structure and mitochondrial DNA pattern of Libyan chickens were thoroughly described. The comparative genomics analyses with 27 other breeds, including local indigenous types and commercial lines, were conducted using SNP-based genetic distance matrices, D-loop analysis, and mitochondrial DNA sequence alignments. Together, these results give a complete picture of where Libyan chickens fit in the global phylogenetic context of Gallus gallus and define their evolutionary uniqueness (Wen et al., 2025).

The Libyan chickens were genetically divergent, had intermediate ancestry with Asian breeds, and had a unique mitochondrial genome organization. These characteristics have emphasized the genetic worth of Libyan chickens and their contributions to biodiversity conservation and poultry genetics. It is important to conserve the Libyan breed to ensure the continuation of desirable traits such as disease resistance and environmental adaptability. It is therefore recommended to conserve and breed Libyan chickens to ensure the sustainability of their genetic traits and improve their productivity. Further studies with larger regional samples and the creation of genomic databases are crucial for the conservation, sustainable utilization, and inclusion of Libyan chickens in the global poultry genetic resource pool.

We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.

This research has been supported by the Universitas Diponegoro. The authors would like to provide their sincere thanks to the farmers and agricultural officers for their cooperation and support while collecting data. The technical staff and research team are also thanked specifically for their support throughout the research.

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