Modern-day chicken breeds mostly evolved from Red Jungle fowl (RJF), which is evident from archeological discoveries (Darwin, 1868; Beebe, 1918; Danforth, 1958; Morejohn, 1968; Fumihito et al., 1994). There are also some reports on the contribution of other Jungle fowl in evolving many breeds across the Globe (Eriksson et al., 2008; Lawal, 2017). However, according to the available data, it is unclear when and where the first domestication of chickens took place (Zeuner, 1963; Crawford, 1984; West and Zhou, 1988; Fumihito et al., 1996; Liu et al., 2006; Xiang et al., 2014; Peters et al., 2015). In early 3,200 BC, chicken domestication was observed in the Indus valley, and accepted as the epicenter of chicken domestication (Zeuner, 1963). Wild RJF can be found in the forests of South East Asia and India; when people domesticated the chicken and spread it to different parts of the world, the genome landscape of domestic chickens was molded by natural and artificial selection, bringing about a wide range of breeds and ecotypes (Liu et al., 2006; Stevens, 1991; Peterson and Brisbin, 1999; Silva et al., 2009; Gifford-Gonzalez and Hanotte, 2011; Mwacharo et al., 2011). The domestic chicken has broad phenotypic variations. However, the RJF lack the phenotypic variations that would be the result of domestication, for example, plumage color and other morphological characteristics, behavioral and production traits, adaptation to different agro-ecosystems, and rigid human selection for production as well as aesthetic qualities (Schütz et al., 2001; Keeling et al., 2004; Tixier-Boichard et al., 2011). Following domestication, large-scale breeding programs have resulted in more than sixty chicken breeds representing four particular genealogies: egg-type, game, meat-type, and bantamweight (Moiseyeva et al., 2003). According to the taxonomy, the genus Gallus is composed of four species: G. gallus (Red Jungle Fowl), G. lafayettei (Lafayette’s Jungle Fowl), G. varius (Green Jungle Fowl), and G. sonneratii (Grey Jungle Fowl). At present, RJF has five sub-species based on phenotypic traits and geographic distribution of the populations: G. g. gallus (South East Asia RJF), G. g. spadiceus, G. g. bankiva, G. g. murghi (Indian RJF) and G. g. jabouillei (Niu et al., 2002). The domestic chicken is considered either a subspecies of RJF (G. g. domesticus) or a separate species, G. domesticus. Scientists are concerned about the genetic integrity and conservation status of the wild RJF and those held in avicultural assortments. It was revealed that the domestic chicken is hybridized with the wild RJF, resulting in the erosion of the genetic purity of the wild birds (Peterson and Brisbin, 1999; Brisbin et al., 2002; Nishibori et al., 2005). Because the previous examinations depend on phenotypic characters, small samples were utilized for DNA investigations. Mitochondrial D-loop sequence phylogeny and nuclear gene analyses demonstrated conceivable hybridization between GJF-RJF/domestic birds (Nishibori et al., 2005). Based on these reports, the evaluation of the genetic uniqueness of Indian RJFs is significant for conservation and population studies.

In the study of chicken hereditary diversity, microsatellites have been effectively utilized (Karsli and Balcıoğlu, 2019). For examining the hereditary connections between chicken populations, hereditary assorted diversity estimates utilizing the exceptionally polymorphic variable number of tandem repeat loci have yielded reliable and precise data. Over the previous decade, the utilization of maternally inherited mitochondrial DNA (mtDNA), particularly its complete displacement-loop (D-loop) region, has expanded. To track genetic information about chicken ancestral breeds, demonstrating the phylogenetic relationship, genetic distance, and variability within and between populations, one of the most significant and remarkable molecular tools, the nucleotide sequence of the mitochondrial D-loop region, is used (Moore, 1995; Nishibori, 2004). The mtDNA or an explicit part of mtDNA (e.g. D-loop) sequencing gives precise data on evolution and hereditary diversity (Fumihito et al., 1994; Di Lorenzo et al., 2015). The D-loop region evolves much faster than different areas of the mtDNA and does not encode a protein. A variation study was done utilizing the mtDNA D-loop region and HVI domain of 397 bp fragments for 398 African native chickens from 12 countries; 12.59% polymorphic sites were discovered (Mobegi, 2006). Likewise, 25 individuals from six native Chinese chicken populations recorded polymorphic variation rates between the range of 7.05% and 5.54%, respectively (Niu et al., 2002; Liu et al., 2004). However, there are no reports on Indian native chickens at the mitochondrial genomic level. Examining native chickens at the mitochondrial genomic level can explain the mitochondrial genomic premise of their disparities and the particular attributes of indigenous chickens can be accurately investigated. The comprehension of phylogeography will clarify the demographic history, origin, and extension of chicken species. To defeat the issue of parallel mutations and lineage exchange between different populaces, network analysis has enhanced phylogenetic trees (Bandelt et al., 1999). India is a huge nation that contains a unique scope of altitudes and climates, meaning native chickens have an astounding genetic diversity. As indicated by ICAR-NBAGR, India has 19 indigenous breeds: Ankaleshwar, Aseel, Busra, Chittagong (Malay), Danki, Daothigir, Ghagus, Harringhata Black, Kadaknath, Kalasthi, Kashmir Favorolla, Miri, Nicobari, Punjab Brown, Tellichery, Mewari, Kaunayen chicken, Hansli, and Uttara (Mogilicherla et al., 2022). There is a need to characterize native chicken lines at the molecular level to enact protection and improvement activities to benefit the nation. With the extended focus on genetic preservation, remarkable alleles may be helpful when making choices to keep up with native varieties. Therefore, the current examination aims to assess the hereditary divergence between twenty-two native Asian breeds and seven native Indian chicken breeds (i.e. Aseel, Ghagus, Nicobari Brown, Tellicherry, Kadaknath, Haringhata Black, and Red Jungle Fowl) utilizing mtDNA NGS sequence data.

The proposed whole mtDNA sequence analysis was effectively used to characterize the seven indigenous Indian chickens along with twenty-two native Asian chickens. The blood samples were collected and mtDNA was extracted from seven Indian native chicken breeds: Aseel, Ghagus, Nicobari Brown, Tellichery, Kadaknath, Haringhata Black, and Red Jungle Fowl. We successfully amplified mtDNA using an amplification method and NGS sequencing was done using an Ion PGM™ sequencer at Genotypic Technology’s Genomics facility. We obtained 757073, 89859, 105503, 50617, 15764, 264309, and 36685 sequencing reads from seven Indian native chickens, respectively (Table 4; Supplementary Table S1). The assembled complete mitochondrial genomes for seven Indian native chickens were submitted to Genbank under accession numbers, KP211418.1, KP211419.1, KP211422.1, KP211424.1, KP211425.1, KP211420.1, and KP211423.1 for Aseel, Ghagus, Nicobari Brown, Tellichery, Kadaknath, Haringhata Black, and Indian Red Jungle Fowl, respectively.

The complete length of mtDNA of Indian local chickens was 16,775 bp. Like most vertebrates, it contains a common structure, including 2 rRNA genes, 22 tRNA genes, 12 protein-coding genes, and 1 D-loop region (Lin et al., 2016; Gu and Li, 2020). The four nucleotides’ (i.e., A, T, G, and C) overall composition of assessed mtDNA was 30.26%, 23.76%, 32.48%, and 13.50%, in the order G > A > T > C, respectively. The inception codons for all the coding proteins was ATG, aside from COX1 which is GTG (Table 5). The heavy (H) strand of mtDNA encoded all the mtDNA genes and the light (L) strand encoded four sorts of tRNA genes and ND6 genes. Every one of these genes had 15 spaces in the length of 2–227 bp and had 3 overlaps in the length of 1-8 bp. These genes had three sorts of termination codons, namely TAA, TAG, and TGA. The “T– – “ is the 5′terminal of the adjoining gene (Anderson et al., 1981). The lengths of the two rRNA genes were 976 bp and 1621 bp. Among 12 protein-coding genes, the longest one was the ND5 gene (1818 bp) and the most limited one was the ND4L gene (297 bp). As seen in other types of chicken, four tRNA genes were circulated in protein-coding genes, varying from 66 to 135 bp in size (Yu et al., 2016; Liu et al., 2016a; Liu et al., 2016b; Lin et al., 2019). The D-loop region was situated among ND6 and rRNA with a length of 1227 bp (Table 5; Supplementary Table S2).

It is hypothesized that chickens (Gallus gallus domesticus) were domesticated from wild jungle fowls in Southeast Asia approximately 10,000 years ago (Ahmed et al., 2017). Native fowl extinction can be improved by modifying cultivation, taking care of the chickens, and better prosperity spread. However, selection and crossbreeding can be used for genetic improvement. Aside from conventional breeding, molecular breeding techniques (Microsatellite markers and SNPs), functional genomics, gene silencing, and genome editing techniques can be used to improve the quality of traits in chickens. Microsatellite markers, SSCP, and sequencing techniques can be used to distinguish proof of polymorphism on genes and assessment of the impact of polymorphism on growth traits in chickens. Molecular genetics has given a few useful assets to exploit the extraordinary abundance of polymorphism at the DNA level, for example, DNA-based genetic markers. The advantage of mtDNA is that it is present in higher copy numbers within the cells and thus can be recovered even from highly degraded specimens (Fumihito et al., 1996). For species identification, forensic studies, and anthropological and evolutionary research, the mitochondrial genome SNPs are assuming a significant role (Yacoub and Fathi, 2013). In 2016, single nucleotide polymorphism was observed, and eleven SNPs in the D-loop region of chicken mitochondrial DNA were reported (Jia et al., 2016).

To set up a phylogenetic relationship, studies were conducted on domestic fowl, different RJF subspecies, and other jungle fowls. In 1994, an investigation proposed that G.g. gallus is a major or sole contributor to a single domestication event, and three further species of RJF mtDNA D-loop region sequences were taken for phylogenetic analysis. This proved G.g. gallus is the real matriarchic origin of all the domestic poultry (Fumihito et al., 1994; Fumihito et al., 1996). In 2009, two ribosomal genes (12S rRNA and 16S rRNA) were used to study the genetic divergence between Indian RJF (G.g. murghi), Gallus gallus subspecies (G.g. spadicus, G.g. gallus, and G.g. bankiva) including G.g. domesticus (domestic fowl), and three other Gallus species (G. varius, G. lafayetei, and G. sonneratii) and found that, compared to other jungle fowls, G.g. murghi was closer to Gallus gallus subspecies, however between Indian RJF and other RJF subspecies, the divergence was very low (Gupta et al., 2009). Furthermore, the study found no noticeable contrasts between G.g. spadicus and G.g. gallus yet G.g. bankiva showed contrasts with both the genetic and phylogenetic relationships among these species (Ulfah et al., 2016). Recent studies confirmed multiple domestications of Indian and other domestic chickens and provided evidence for the domestication of Indian birds from G.g. spadiceus, G.g. gallus, and G.g. murghi (Kanginakudru et al., 2008; Mekchay et al., 2014; Maw et al., 2015). These studies avoided the two different subspecies of red jungle fowl i.e. G.g. murghi and G.g. jabouillei, however, they provided a framework for genetic studies in wild jungle fowls and native and domestic chicken breeds. Considering solid confirmations of the domestication of chicken in the Indus valley, it might be interesting to contemplate the genetic relatedness between Indian RJF and other RJF subspecies including G.g. domesticus and other jungle fowls (Kawabe et al., 2014).

Indian RJFs are widely distributed across 51 × 10⁵ km² in 21 states of India (Fernandes et al., 2009; Mogilicherla et al., 2022) (Figure 1). We were more interested to explore the phylogenetic relationship of Indian RJF with reference RJF and native Asian breeds along with other native Indian breeds and understand the contribution of Indian RJF, G. g. murghi, to the domestication event. Hence, in the present study, seven native Indian breeds (Aseel, Ghagus, Nicobari brown, Kadaknath, Tellicherry, H. black, and Indian Red Jungle fowl) and twenty-two native Asian breeds were used to study the nucleotide sequence variation in complete mtDNA and the most variable region of mtDNA D-loop region to build up the phylogenetic relationship among the Indian breeds (Table 1; Table 2). Currently, no sequence data is accessible from these seven native Indian breeds. However, one of these breeds is likely to have been a contributor to one of the earliest known chicken domestication events, for example in the Mohanjo-Daro Indus valley.

The preserved traits like Struthioniformes, Falconiformes, and Sphenisciformes have been depicted across a variety of avian species (Haring et al., 2001; Wani et al., 2014). The Indian indigenous chicken D-loop area sequence has cytosines and guanine strings close to each other, making the advancement of a consistent hairpin structure possible (Quinn and Wilson, 1993). In all neighborhood chickens of India, the conserved sequence motifs of TACAT and TATAT were found. Such subjects are depicted as termination-associated sequence segments (TASs) with mtDNA synthesis (Foran et al., 1988). Both Galliformes and mammals have TASs, which may propose a strong auxiliary capacity of the D-loop area of the two genera, while the lack of variety in TASs among the Galliformes may be a direct result of the particular utilitarian constraints. The eight haplotypes recognized from the Indian indigenous chicken and the phylogenetic examination spoke to the formative associations.

A large number of the investigations on domestic chicken origins have focused on the D-Loop; there were additionally a few examinations concentrating on mitochondrial genomics to analyze domestic chicken origins (Miao et al., 2013). Kauai feral chicken mitochondrial phylogenies (D-loop and whole mtDNA) revealed two different clades within their samples (Gering et al., 2015). Maybe the reason for the difference was that the samples for the examination were different between the whole mtDNA phylogeny and mtDNA D-loop phylogeny. We found that the mitochondrial phylogenies (D-loop and whole Mt genome) uncovered a few clades, and their examination re-evaluated the worldwide mtDNA profiles of chickens and encouraged our study of the settlement in India.

From the mtDNA examination, we saw that Indian RJF is the origin of reference RJF as well as Indian breeds (i.e. Aseel, Ghagus, Nicobari brown, Kadaknath, H. black, and Tellicherry). The reference RJF is closely related to the Indian reference RJF, which, in turn, has contributed to the genetic makeup of six native Indian breeds. This is likewise true for the Indian chicken, which originated from independent domestication from Indian RJF and likely from other RJF species. Curiously, the mixing of various Indian breeds with Indian RJF could explain how the current-day Asiatic chicken may have originated from various ancestors through numerous domestication events and such multi-origin breeds could still be seen in a single geographical location. This is in agreement with the current-day perceptions of native Japanese chickens that originated from various regions (Oka et al., 2007). However, all the breeds aside from Indian RJF form a solitary group, suggesting a common ancestor for these birds, including jungle fowls and domestic birds. The separation of the Indian RJF from the main group of breeds shows the possibility of a speciation event. Our examination uncovered that the Indian breeds are relatively pure with uncommon hybridization with reference RJF. In the current investigation of Indian breeds, we did not come across recognizable hybridization at least in the recent past, as shown by a clear separation of Indian RJF clades from reference RJF in mtDNA-based phylogeny. All these results indicate the genetic integrity of the Indian RJF.

In high-altitude birds, hypoxia is an unavoidable environmental stress; in natural selection, every step of aerobic respiration had to be experienced to improve the adaptability to hypoxia (Altshuler and Dudley, 2006; Scott, 2011; Scott and Milsom, 2006). Mitochondria play an important role in aerobic respiration through oxidative phosphorylation, because the majority of produced cell ATP is consumed for cellular oxygen uptake (Chandel and Schumacker, 2000; Xu et al., 2007). To determine the role of mitochondrial genes in high-altitude adaptation, six high-altitude Phasianidae birds and 16 low-altitude relatives’ mitochondrial genomes were analyzed, and four lineages were found for this high-altitude habitat (Zhou et al., 2014). Their results strongly suggest that the adaptive evolution of mitochondrial genes, i.e. ND2, ND4, and ATP6, played a critical role during the independent acclimatization to high altitude by galliform birds. In Tibet, a chicken breed was found to have a missense mutation in the MT-ND5 subunit of the NADH dehydrogenase gene for high-altitude adaptation (Bao et al., 2007). To explore the regulatory mechanisms for hypoxia adaptability, the ATP-6 gene was sequenced from 28 Tibetan chickens and 29 Chinese domestic chickens; six SNPs were detected (Zhao et al., 2015). In high-altitude adaption, cytochrome c oxidase (COX) was the key mitochondrial gene and plays an important role in oxidative phosphorylation regulation and oxygen sensing transfer. For identifying the COX gene SNP, the Tibet Chicken and four lowland chicken breeds (Dongxiang Chicken, Silky Chicken, Hubbard ISA White broiler, and Leghorn layer) were used and 13 haplotypes were defined for the 14 SNPs. It was concluded that the significant difference in MT-CO3 gene mutation might have a relationship with the high-altitude adaptation (Bao et al., 2008). MT-CO3 gene was sequenced by using 125 Tibetan chickens and 144 Chinese domestic chickens; eight SNPs were identified, and were defined into nine haplotypes. They found positive and negative haplotype associations with high-altitude adaptation (Sun et al., 2013). However, the MT-COI gene was sequenced from 29 Tibetan chickens and 30 Chinese domestic chickens, and nine SNPs were detected (Zhao et al., 2015). In our study, ND3, ND4, ND5, ATP6, COX1, and COX2 showed high-altitude lineages between Nicobari brown and Reference RJF birds which may help chickens to evolve to adapt to Nicobari Island environments (Figures 5–8).

mtDNA mutations contribute to enclosing both tissue-specific and multiple-system disorders in human diseases (Taylor and Turnbull, 2005; Tuppen et al., 2010). The spindle-associated chromosomal exchange did not show antagonistic consequences for fertilization on subsequent embryo/fetal development in the rhesus monkey (Tachibana et al., 2009). Subsequently, this method may speak to another dependable restorative way to deal with the transmission of mtDNA mutations in influenced families. Mitochondrial heterogeneity is the presence of at least two kinds of mitochondrial (mt) DNA in the same individual/tissue/cell and it is firmly related to animal health and disease. In mtDNA, ND2 is a protein-coding gene and it partakes in the mitochondrial respiratory chain and oxidative phosphorylation. In cloned sequencing of the ND2 region, numerous potential heteroplasmic locales were recognized, which possibly reflected bountiful heteroplasmy in the chicken mitochondrial genome (Yang et al., 2020). These outcomes give a significant reference for further research on heteroplasmy in chicken mitochondria. Recently, an examination utilized complete mtDNA from tuberculosis patients’ blood samples and explored the conceivable mtDNA variations (Tonsing et al., 2020). Twenty-eight non-synonymous variants were found and most of the variations lie in the D-loop of the non-protein-coding region of the mitochondrial DNA. Runting and stunting syndrome (RSS) generally happens early in life and causes low body weight and extensive economic losses in the commercial broiler industry (Kang et al., 2012). In sex-linked dwarf (SLD) chickens, the RSS is related to mitochondria dysfunction, and mutations in the TWNK gene are one reason for mtDNA exhaustion (Li et al., 2019; Hu et al., 2020). We recommend that mutations in the mitochondrial genome should be validated further to fully understand their relationship with animal diseases.

Deep sampling using the NGS technique offers a straightforward, high-throughput, and cost-effective platform for effectively detecting and measuring mitochondrial heteroplasmy in complete mitochondrial genomes. The main limitation of this research is that only seven native Indian breeds were used. Nevertheless, the information that was collected will be useful in finding heteroplasmy in various chicken breeds both domestically and internationally.

In summary, the eight haplotypes of the native chicken population showed a relatively rich genetic pool, and molecular information on genetic diversity revealed may help in developing genetic improvement and conservation strategies to better utilize precious genetic reserves. The phylogenetic relationship of native Indian chicken breeds using entire mtDNA and D-loop sequences showed that the South East Asian RJF is distant from the Indian RJF but genetically close to the Indian breeds, whereas the Indian Aseel breed was more closely related to the reference RJF than the Indian RJF. The grouping of Indian RJF separated from Indian native chickens and the presence of subcontinent explicit haplogroups gives additional proof for an independent domestication event of chickens in the subcontinent. For high-altitude hypoxic adaptation, it is important to improve the efficiency of oxygen usage instead of enhancing oxygen uptake and transport. Thus, in natural selection, the mitochondrial genome encoded 12 essential structural genes (6 NADH dehydrogenase genes, cytochrome b subunit, 3 cytochrome c oxidase, and ATP synthase subunit), which must have mutated during adaptation to high-altitude hypoxic conditions.