Comparative mitochondrial genomics of the Peruvian Creole sheep (Ovis aries)

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This study analyzes the complete mitochondrial genome of five Peruvian Creole sheep (Ovis aries) individuals to assess their genetic variability and phylogenetic relationships. The mitochondrial genome exhibits a length of 16,617 base pairs (bp) with a guanine-cytosine (GC) content of 39%, encoding 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. Codon usage bias analysis reveals a marked preference for specific codons, particularly in Arginine (Arg), Serine (Ser), and Threonine (Thr), suggesting the influence of mutational pressure on mitochondrial gene evolution. Phylogenetic analysis places the PCS individuals within Clade III, alongside sheep breeds from Russia, Brazil, and New Zealand, but without forming a monophyletic group. The absence of monophyly suggests multiple historical introduction events and potential gene flow with different ovine populations, paralleling patterns observed in Mediterranean and African sheep breeds. The phylogenetic relationship of Peruvian Creole sheep with Brazilian and Chinese lineages highlights the complexity of their genetic background, indicating potential admixture and historical introgression. These findings underscore the need for further genomic research to clarify Peruvian Creole sheep's evolutionary history and support conservation efforts of their zoogenetic resources. Biological sciences/Genetics/Evolutionary biology Biological sciences/Genetics/Genomics Biological sciences/Genetics/Animal breeding Livestock Genomics NGS Andes Zoogenetic resources Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The domestic sheep ( Ovis aries ) is one of the first species domesticated by humans, playing a crucial role in developing agriculture and the economy in various cultures [ 1 ]. Its domestication originated in the Fertile Crescent in the Middle East and spread to Europe, Asia, and Africa. In Latin America, the introduction of sheep occurred during European colonization, adapting to various ecosystems and giving rise to various breeds and local ecotypes [ 2 ]. In countries such as Brazil, Argentina, Peru, Bolivia, Mexico, and Uruguay, sheep farming is an activity of great economic and cultural relevance, especially in indigenous and rural communities [ 3 , 4 ]. It is estimated that the sheep population in Latin America is approximately 80 million head, with a notable presence of creole breeds adapted to local conditions [ 5 ]. In Peru, sheep farming has a long tradition, with ecotypes such as the "criollo serrano" and the "criollo cholo de Piura," as well as the “Junín” breed developed in the country [ 6 ]. Recent studies have evaluated the genetic diversity of these populations. For example, in the Pasco region, the genetic variability of the creole sheep was analyzed using 11 molecular markers, revealing significant genetic diversity [ 7 ]. Similarly, research in Huancavelica has determined the genetic variability and estimated the degree of inbreeding in local creole sheep [ 8 ]. Genetic diversity in sheep populations is essential for the development of productive traits and adaptation to different environments [ 9 ]. The analysis of the complete mitochondrial genome has allowed for a deeper understanding of the evolutionary history and domestication processes of sheep [ 10 ]. Studies in Argentina have genetically characterized populations of creole sheep using the D-loop region of mitochondrial DNA, revealing significant variability and a relationship with Asian haplogroups, suggesting an influence of Spanish breeds introduced during colonization [ 11 ]. Even though Peruvian Creole sheep represents cultural and economic importance in the country, very little is known about its genetics. Here, we sequenced, assembled, and annotated the complete mitochondrial genome of five individuals that belong to the Peruvian Creole Sheep. This approach will deepen our understanding of the evolutionary history and adaptation processes of these sheep populations in the Peruvian context, promoting its efficient conservation and sustainable use for modern breeding programs. Materials and Methods Sampling and Sequencing Five samples of sheep identified as Creole by a technical assistant and owners from Huayllay district (4,340 m.a.s.l.), Pasco region were collected. The samples were taken from the ear of each individual with a Tissue Sampling Unit (TSU) for DNA Collection (Allflex, Merck & Co. Inc., Rahway, NJ, USA). Each sample was labelled and data from the owners was registered. All tissue samples were outsourced to Novogene facilities (Novogene Corporation Inc. (Sacramento, CA) for whole genome sequencing (30x-60x). All DNA extraction and library construction work were conducted by the Novogene Team. Assembly and Annotation of the Mitogenomes The mitochondrial genomes of Ovis aries from New Zealand (SRR19144868, SRR19144759, and SRR19144905) and Brazil (SRR501837, SRR501855, and SRR501846) were assembled and annotated in this study (Table S1 ). Adapters and low-quality reads were removed using the default settings of TrimGalore v0.6.7 [ 12 ] and Trimmomatic v0.36 software. The trimmed sequences were then used for mitochondrial genome assembly through the GetOrganelle pipeline [ 13 ], which integrates multiple tools, including SPAdes v3.11.1 [ 14 ], Bowtie2 v2.4.2 [ 15 ], and BLAST + v2.11 [ 16 ]. Annotations for protein-coding genes, transfer RNAs (tRNAs), and ribosomal RNA (rRNA) genes within the mitochondrial genome were performed using the automated mitochondrial gene annotation tools available in Geseq, hosted on the CHLOROBOX web service [ 17 ]. The termination codon was omitted, and the 13 protein-coding genes (PCGs) were subsequently concatenated using the Concatenate Sequence Alignment function. Codon usage patterns were analyzed through the relative synonymous codon usage (RSCU) function in MEGA v11 [ 18 ]. A graphical depiction of the circular mitochondrial genome was generated using OGDRAW v1.3.1 [ 19 ]. Sliding window analysis was performed on the aligned, complete mt genome sequences of the O. aries using DnaSP v.5 [ 20 ]. Phylogenetic Analysis To determine the genetic affiliation of the Peruvian Creole sheep, a total of 57 mitochondrial genomes from other Ovis species available in GenBank were analyzed, incorporating Capra hircus as an outgroup due to its taxonomic classification within the same subfamily Caprinae (Table S1 ). Genomic alignments were performed using MAFFT v7.475 [ 21 ], and phylogenetic relationships were inferred through a maximum likelihood (ML) approach, employing the GTR + GAMMA evolutionary model. The robustness of the tree was assessed through 1000 nonparametric bootstrap replicates using RAxML v8.2.11 [ 22 ], and the resulting phylogenetic trees were visualized with iTOL [ 23 ]. Results Genome Size and Organization The complete mitochondrial genome of the Peruvian Creole has a length of 16,617 base pairs (bp) and a guanine-cytosine (GC) content of 39%, with no variations between B1, B2, B3, B4 and B5 (Fig. 1 , Fig. S1 ). The genomic sequence includes 13 protein-coding genes (ND1–ND6, ND4L, COX1–COX3, CYTB, ATP6, and ATP8), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes (12S rRNA and 16S rRNA), and the control region (loop D). The percentages of nitrogenous bases were adenine (A) 33.66%, thymine (T) 27.40%, cytosine (C) 25.81%, and guanine (G) 13.12%. Codon Usage and Sliding Windows The analysis of relative synonymous codon usage (RSCU) revealed differentiated patterns of codon preference among the amino acids evaluated. Amino acids such as Arginine (Arg), Serine (Ser), and Threonine (Thr) showed a marked bias towards certain specific codons, evidenced by RSCU values greater than 4. This indicates a significant preference in the coding of these amino acids. In contrast, amino acids such as Methionine (Met) and Tryptophan (Trp), encoded by a single codon, did not present variation in RSCU (Fig. 2 ). The mitochondrial genomes of Peruvian Creole sheep (B1–B5) exhibited identical characteristics in gene length and A + T content (Table 2 ). The total genome length was 16,617 bp, with an average A + T content of 59.6%. Coding regions maintained a consistent nucleotide composition, with Nd1 (959 bp, 59.54% A + T), Cox1 (1545 bp, 59.55% A + T), and Nd5 (1821 bp, 61.12% A + T) displaying structural homogeneity. Among ATP synthase genes, Atp8 presented the highest A + T content (69.15%), while CytB retained a moderate value (58.42%). The analysis of nucleotide diversity (π) across the genomic sequence revealed distinct variation patterns at different loci. Notable diversity peaks were observed, with π values approaching 0.004 in the regions associated with the ND1, ND4, and ND5 genes (Fig. 3 ). Table 2 Characterization of protein-coding genes in the mitochondrial genomes of Peruvian Creole sheep. The values presented correspond to the five mitochondrial genomes of PCS (B1–B5), which present identical characteristics in gene length and A + T content. Gene Gene Length (bp) A + T Content (%) Protein Length (aa) Nd1 959 59.54 319 Nd2 982 63.54 327 Cox1 1545 59.55 515 Cox2 684 62.43 228 Atp8 201 69.15 67 Atp6 680 60.26 226 Cox3 784 54.85 261 Nd3 347 58.21 115 Nd4L 297 60.27 99 Nd4 1378 60.96 459 Nd5 1821 61.12 607 Nd6 528 64.77 176 CytB 1140 58.42 380 Phylogenetic Analysis The phylogenetic analysis based on mitochondrial sequences revealed the genetic structure of different populations of Ovis aries and their close relatives. The phylogenetic tree, constructed using the maximum likelihood method, showed three main clades with high bootstrap support in most branches (Fig. 4 ). Representative sequences from various breeds and lineages of O. aries from different regions of the world were included, as well as related species such as O. ammon and O. orientalis , which are grouped into well-defined clades. The mitochondrial genomes of interest, corresponding to Peruvian samples (B1, B2, B3, B4, and B5), belonging to Criollo sheep, clustered within Clade III, specifically in Subclade III, along with sheep breeds from Russia, New Zealand, and Brazil. However, these Peruvian samples do not form a monophyletic clade but are dispersed within the subclade, suggesting that they may have distinct mitochondrial origins. Samples B1, B2, B3, and B4 formed a well-defined group with 90% BS support and exhibited a closer phylogenetic relationship with Criollo sheep sequences from Brazil, indicating a possible historical connection between these lineages. In contrast, sample B5 clusters more closely with a Yemule sheep from China. Clade I includes the wild lineages of O. ammon and O. orientalis , including the Argali and Asiatic Mouflon variants. In contrast, Clade II consists predominantly of domestic sheep breeds from China, such as the Duoma, Guide, and Qiaoke breeds. Clade III exhibited greater geographical diversity, with sheep breeds from Russia, New Zealand, Brazil, and Peru. Discussion The analysis of relative synonymous codon usage (RSCU) in the mitochondrial genome of the PCS suggests distinct codon preferences, particularly in amino acids such as Arginine (Arg), Serine (Ser), and Threonine (Thr), which exhibited a marked bias toward specific codons (RSCU > 4). This pattern appears to align with previous studies indicating that codon usage bias (CUB) may be shaped by a combination of mutational pressure, natural selection, and genetic drift, with selection potentially acting on translation efficiency and accuracy in mitochondrial genes. The preference for certain synonymous codons has been documented across various taxa, including fishes, birds, and mammals, where overrepresented codons vary by species [ 24 ]. However, given the high mutation rate characteristic of mitochondrial genomes, it is plausible that mutational pressure plays a predominant role in shaping codon preferences in Ovis aries , rather than selection for translational efficiency [ 25 ]. Nucleotide diversity (π) is a measure of genetic variation within a population, indicating the average differences between pairs of DNA sequences. Certain genes exhibit higher nucleotide diversity in mitochondrial genomes, reflecting their evolutionary dynamics. Studies have identified significant variability in genes such as ND1, ND2, ND4, and ND5, which are part of the mitochondrial respiratory chain complex I. For instance, previous research reported multiple nucleotide substitutions in the ND1 gene, including both synonymous and missense mutations, highlighting its polymorphic nature. Similarly, the ND2, ND4, and ND5 genes have been observed to harbor numerous mutations, contributing to the overall genetic diversity of mitochondrial DNA [ 26 ]. These variations can influence mitochondrial function and have been associated with various diseases. The observed peaks in nucleotide diversity within the ND1, ND4, and ND5 regions suggest that these loci are subject to higher mutation rates or selective pressures, underscoring their importance in studies of mitochondrial genetics and evolution [ 27 , 28 ]. The mitochondrial diversity observed in Peruvian Creole sheep reflects a complex pattern of genetic structuring, characterized by the absence of a monophyletic clade, suggesting multiple introduction events and gene flow with other ovine populations. This phenomenon is consistent with reports on African sheep breeds, where high genetic variability and the lack of clear phylogeographic structuring have been attributed to the historical mobility of flocks and transcontinental trade [ 29 ]. Likewise, the phylogenetic relationship between Peruvian and Brazilian sheep samples supports the hypothesis of a shared origin in South America, while the proximity of sample B5 to a Chinese sheep suggests the possibility of migration events or independent introductions. This pattern of genetic diversity is similar to that observed in Mediterranean sheep breeds, where genetic differentiation has been shaped by ancient migration routes and commercial interactions [ 30 ]. The phylogeny of Peruvian Creole sheep, lacking a defined monophyletic grouping, indicates the presence of historical introgression, which may have contributed to the current genetic heterogeneity of these populations. Additionally, the phylogenetic analysis suggests that the genetic diversity in Peruvian Creole sheep may have been influenced by dispersal events like those observed in Central Asian sheep populations. A recent study on ancient sheep mitogenomes from China and Uzbekistan revealed that certain sheep populations acted as centers of genetic diffusion to new regions, facilitating adaptation to diverse environments [ 31 ]. The clustering of Peruvian samples within the same clade without a clear monophyletic structure reinforces the hypothesis that these lineages have undergone hybridization processes with different ovine populations over time. This phylogenetic behavior is consistent with that observed in Mediterranean and Bulgarian breeds, where genetic differentiation has been influenced by multiple dispersal and selection events [ 32 ]. Thus, the population structure present in Peruvian Creole sheep not only reflects a history of multiple introductions but also a possible adaptive convergence resulting from natural and artificial selection in different environments. Conclusions The genetic diversity observed in Peruvian Creole sheep, characterized by the absence of a monophyletic clade, suggests multiple introduction events and historical gene flow with various ovine populations. This pattern aligns with findings in other geographically diverse breeds, where ancient migration routes, trade interactions, and human-driven selection have shaped genetic differentiation. The clustering of Peruvian samples within the same clade without a defined monophyletic structure indicates that these lineages have undergone hybridization with different populations over time. This underscores the importance of considering not only maternal lineages but also historical and anthropogenic factors when studying genetic diversity and conservation strategies. Further research, incorporating larger datasets and complementary genomic approaches, is essential to fully elucidate the evolutionary history and adaptive mechanisms of these populations, ensuring the preservation of their genetic resources. Declarations Data availability The dataset supporting the conclusions of this article is available in the NCBI repository (BioProject: XXXX). Received: ; Accepted: Published online: Acknowledgments We express our special appreciation and thanks to the owners of the PCS individuals located in district of Huayllay (Pasco, Peru) who kindly provided samples of their individuals for this work. We also thank the Comunidad Campesina de Huayllay for supporting the logistic activities for field work. Finally, C.I.A. thanks Vicerrectorado de Investigación of UNTRM, and the Bioinformatics High-Performance Computing server of Universidad Nacional Agraria la Molina for providing resources for data analysis. Author contributions J.C. and C.I.A. conceived the study; F.-A.C., T.B.Y.-H. and C.I.A. planned and designed the field work; F.-A.C., T.B.Y.-H. D.F. and C.I.A. managed the animals and organized the collection of the samples; F.-A.C., T.B.Y.-H. and D.F. performed the experiments; R.E., Y.R., P.C., R.H.-Q. and D.F. performed bioinformatic analysis; J.C. and C.I.A. were responsible of funding acquisition; F.-A.C., T.B.Y.-H., J.C. and C.I.A. were in charge of project administration. R.E., Y.R. and D.F. wrote the original draft; All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the following two research projects: “Creación del servicio de agricultura de precisión en los departamentos de Lambayeque, Huancavelica, Ucayali y San Martín 4 departamentos-AGPRES” and “Mejoramiento de los servicios de investigación y transferencia de tecnologías para el manejo sostenible de la ganadería caprina en bosque seco y costa central en los departamentos de Tumbes, Piura, Lambayeque, Amazonas, La Libertad, Áncash y Lima-PROCAP” of the Ministry of Agrarian Development and Irrigation (MIDAGRI) of the Peruvian Government, with grant numbers CUI 2449640 and CUI 2506684, respectively. Competing interests The authors declare no conflicts of interest. Ethics approval and consent to participate The sample collection from the cattle specimen was conducted in accordance with the Peruvian National Law No. 30407: “Animal Protection and Welfare”. References Daly, K. G. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6323309","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":438535541,"identity":"b12dcce0-6ad1-4438-a553-1bbb72cb7140","order_by":0,"name":"Richard Estrada","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Estrada","suffix":""},{"id":438535542,"identity":"7626590a-9e9a-4e7a-8db3-f5f165eba92e","order_by":1,"name":"Yolanda Romero","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Yolanda","middleName":"","lastName":"Romero","suffix":""},{"id":438535543,"identity":"5ae69343-7553-4e86-b428-a1d90a8cc863","order_by":2,"name":"Deyanira Figueroa","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Deyanira","middleName":"","lastName":"Figueroa","suffix":""},{"id":438535544,"identity":"e5853f20-1bfc-4d9a-a0ea-cf02a4190701","order_by":3,"name":"Flor-Anita Corredor","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Flor-Anita","middleName":"","lastName":"Corredor","suffix":""},{"id":438535545,"identity":"cec8662f-2acd-47bf-b83d-772527abb984","order_by":4,"name":"Teodoro B. Yalli-Huamani","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Teodoro","middleName":"B.","lastName":"Yalli-Huamani","suffix":""},{"id":438535546,"identity":"5f09a82f-3fd8-4f35-b4b9-ef7b040cd99b","order_by":5,"name":"Pedro Coila","email":"","orcid":"","institution":"Universidad Nacional del Altiplano de Puno","correspondingAuthor":false,"prefix":"","firstName":"Pedro","middleName":"","lastName":"Coila","suffix":""},{"id":438535547,"identity":"1a435beb-89d2-4a33-ba2d-0bbc8c7b1772","order_by":6,"name":"Renan Hañari-Quispe","email":"","orcid":"","institution":"Universidad Nacional del Altiplano de Puno","correspondingAuthor":false,"prefix":"","firstName":"Renan","middleName":"","lastName":"Hañari-Quispe","suffix":""},{"id":438535548,"identity":"93cd1185-474f-42a6-9962-1c240571a388","order_by":7,"name":"Juancarlos Cruz","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria (INIA)","correspondingAuthor":false,"prefix":"","firstName":"Juancarlos","middleName":"","lastName":"Cruz","suffix":""},{"id":438535549,"identity":"f425ef47-e78a-4797-b13b-cf4401a48b07","order_by":8,"name":"Carlos I. Arbizu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYHACxgNgSoKxgYGhAibIhl8PkpYzDAw8JGgBWdhGhBb+9rMPDnz4wyBvPru5+TPvvMN59tKnExg+lB1mMGdvwKpF4ky6wcGZbQyGc+4cbDDm3Xa4mIcvdwPjjHOHGSx7DmDVYsCQxnCYt4GBcYZEYkMyUEtiDw/vBmbetsMMBjcSsGvhf8ZwmOcPgz1Iy2HeOVAtf0Fa7j/ArkUCaAsPG0MiUEtjM28DVAsj2Bbs3pe48YwB6BeJ5BkyB5sZ5xxLT+w5w7vhYM+5dB7LHuwO4+9PY3zw4Y+N7Qzp9scf3tRYJ7b38G588KPMWs6cHbv3YZahckFqeQzwacAOyNAyCkbBKBgFwxMAANm6XzNrREnaAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (UNTRM)","correspondingAuthor":true,"prefix":"","firstName":"Carlos","middleName":"I.","lastName":"Arbizu","suffix":""}],"badges":[],"createdAt":"2025-03-27 21:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6323309/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6323309/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81795528,"identity":"5c8f7080-1c54-4866-8c9d-2718c158fcac","added_by":"auto","created_at":"2025-05-02 03:29:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134069,"visible":true,"origin":"","legend":"\u003cp\u003eThe mitochondrial genome map of the Peruvian Creole sheep, individual B1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/1104d76256a8dee3510a438e.png"},{"id":81795531,"identity":"52d6fbed-a222-4b33-9d51-a3872df86184","added_by":"auto","created_at":"2025-05-02 03:29:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59383,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative synonymous codon usage (RSCU) of the mitochondrial genome’s protein-coding genes of \u003cem\u003eOvis aries\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/5e5f38c66669f2cd209bf9fa.png"},{"id":81795529,"identity":"982b1117-9951-4693-97ae-863ac3511c94","added_by":"auto","created_at":"2025-05-02 03:29:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96187,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of nucleotide diversity (π) in the mitochondrial genome of \u003cem\u003eOvis aries\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/100e8526e74679abffbcd9f0.png"},{"id":81795536,"identity":"32c73f3f-ce37-4a56-b179-7a5073fe9e3e","added_by":"auto","created_at":"2025-05-02 03:30:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":309320,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic tree, constructed using maximum likelihood and based on mitochondrial genomic sequences from \u003cem\u003eOvis\u003c/em\u003e species, displays bootstrap support values exclusively for branches receiving over 70% support. \u003cem\u003eCapra hircus\u003c/em\u003e was designated as the outgroup in this analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/40e4ea50c2c5c374e794596a.png"},{"id":90509776,"identity":"9cd2345d-9784-40fb-93ca-cd6c5c3355c8","added_by":"auto","created_at":"2025-09-03 13:23:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1178715,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/b9a623ed-18a5-405c-87f3-b92de44000db.pdf"},{"id":81795535,"identity":"5e3bb9e1-4be2-4d43-97d2-48693d7738bb","added_by":"auto","created_at":"2025-05-02 03:30:00","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2037311,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-6323309/v1/74b6bdf25049a5cbdcedae5a.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative mitochondrial genomics of the Peruvian Creole sheep (Ovis aries)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe domestic sheep (\u003cem\u003eOvis aries\u003c/em\u003e) is one of the first species domesticated by humans, playing a crucial role in developing agriculture and the economy in various cultures [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its domestication originated in the Fertile Crescent in the Middle East and spread to Europe, Asia, and Africa. In Latin America, the introduction of sheep occurred during European colonization, adapting to various ecosystems and giving rise to various breeds and local ecotypes [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn countries such as Brazil, Argentina, Peru, Bolivia, Mexico, and Uruguay, sheep farming is an activity of great economic and cultural relevance, especially in indigenous and rural communities [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. It is estimated that the sheep population in Latin America is approximately 80\u0026nbsp;million head, with a notable presence of creole breeds adapted to local conditions [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn Peru, sheep farming has a long tradition, with ecotypes such as the \"criollo serrano\" and the \"criollo cholo de Piura,\" as well as the \u0026ldquo;Jun\u0026iacute;n\u0026rdquo; breed developed in the country [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies have evaluated the genetic diversity of these populations. For example, in the Pasco region, the genetic variability of the creole sheep was analyzed using 11 molecular markers, revealing significant genetic diversity [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, research in Huancavelica has determined the genetic variability and estimated the degree of inbreeding in local creole sheep [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eGenetic diversity in sheep populations is essential for the development of productive traits and adaptation to different environments [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. The analysis of the complete mitochondrial genome has allowed for a deeper understanding of the evolutionary history and domestication processes of sheep [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies in Argentina have genetically characterized populations of creole sheep using the D-loop region of mitochondrial DNA, revealing significant variability and a relationship with Asian haplogroups, suggesting an influence of Spanish breeds introduced during colonization [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eEven though Peruvian Creole sheep represents cultural and economic importance in the country, very little is known about its genetics. Here, we sequenced, assembled, and annotated the complete mitochondrial genome of five individuals that belong to the Peruvian Creole Sheep. This approach will deepen our understanding of the evolutionary history and adaptation processes of these sheep populations in the Peruvian context, promoting its efficient conservation and sustainable use for modern breeding programs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eSampling and Sequencing\u003c/h2\u003e \u003cp\u003eFive samples of sheep identified as Creole by a technical assistant and owners from Huayllay district (4,340 m.a.s.l.), Pasco region were collected. The samples were taken from the ear of each individual with a Tissue Sampling Unit (TSU) for DNA Collection (Allflex, Merck \u0026amp; Co. Inc., Rahway, NJ, USA). Each sample was labelled and data from the owners was registered. All tissue samples were outsourced to Novogene facilities (Novogene Corporation Inc. (Sacramento, CA) for whole genome sequencing (30x-60x). All DNA extraction and library construction work were conducted by the Novogene Team.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAssembly and Annotation of the Mitogenomes\u003c/h2\u003e \u003cp\u003eThe mitochondrial genomes of \u003cem\u003eOvis aries\u003c/em\u003e from New Zealand (SRR19144868, SRR19144759, and SRR19144905) and Brazil (SRR501837, SRR501855, and SRR501846) were assembled and annotated in this study (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Adapters and low-quality reads were removed using the default settings of TrimGalore v0.6.7 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Trimmomatic v0.36 software. The trimmed sequences were then used for mitochondrial genome assembly through the GetOrganelle pipeline [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which integrates multiple tools, including SPAdes v3.11.1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Bowtie2 v2.4.2 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and BLAST\u0026thinsp;+\u0026thinsp;v2.11 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Annotations for protein-coding genes, transfer RNAs (tRNAs), and ribosomal RNA (rRNA) genes within the mitochondrial genome were performed using the automated mitochondrial gene annotation tools available in Geseq, hosted on the CHLOROBOX web service [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The termination codon was omitted, and the 13 protein-coding genes (PCGs) were subsequently concatenated using the Concatenate Sequence Alignment function. Codon usage patterns were analyzed through the relative synonymous codon usage (RSCU) function in MEGA v11 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A graphical depiction of the circular mitochondrial genome was generated using OGDRAW v1.3.1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sliding window analysis was performed on the aligned, complete mt genome sequences of the \u003cem\u003eO. aries\u003c/em\u003e using DnaSP v.5 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eTo determine the genetic affiliation of the Peruvian Creole sheep, a total of 57 mitochondrial genomes from other Ovis species available in GenBank were analyzed, incorporating \u003cem\u003eCapra hircus\u003c/em\u003e as an outgroup due to its taxonomic classification within the same subfamily Caprinae (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Genomic alignments were performed using MAFFT v7.475 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and phylogenetic relationships were inferred through a maximum likelihood (ML) approach, employing the GTR\u0026thinsp;+\u0026thinsp;GAMMA evolutionary model. The robustness of the tree was assessed through 1000 nonparametric bootstrap replicates using RAxML v8.2.11 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and the resulting phylogenetic trees were visualized with iTOL [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGenome Size and Organization\u003c/h2\u003e \u003cp\u003eThe complete mitochondrial genome of the Peruvian Creole has a length of 16,617 base pairs (bp) and a guanine-cytosine (GC) content of 39%, with no variations between B1, B2, B3, B4 and B5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The genomic sequence includes 13 protein-coding genes (ND1\u0026ndash;ND6, ND4L, COX1\u0026ndash;COX3, CYTB, ATP6, and ATP8), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes (12S rRNA and 16S rRNA), and the control region (loop D). The percentages of nitrogenous bases were adenine (A) 33.66%, thymine (T) 27.40%, cytosine (C) 25.81%, and guanine (G) 13.12%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCodon Usage and Sliding Windows\u003c/h3\u003e\n\u003cp\u003eThe analysis of relative synonymous codon usage (RSCU) revealed differentiated patterns of codon preference among the amino acids evaluated. Amino acids such as Arginine (Arg), Serine (Ser), and Threonine (Thr) showed a marked bias towards certain specific codons, evidenced by RSCU values greater than 4. This indicates a significant preference in the coding of these amino acids. In contrast, amino acids such as Methionine (Met) and Tryptophan (Trp), encoded by a single codon, did not present variation in RSCU (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mitochondrial genomes of Peruvian Creole sheep (B1\u0026ndash;B5) exhibited identical characteristics in gene length and A\u0026thinsp;+\u0026thinsp;T content (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The total genome length was 16,617 bp, with an average A\u0026thinsp;+\u0026thinsp;T content of 59.6%. Coding regions maintained a consistent nucleotide composition, with Nd1 (959 bp, 59.54% A\u0026thinsp;+\u0026thinsp;T), Cox1 (1545 bp, 59.55% A\u0026thinsp;+\u0026thinsp;T), and Nd5 (1821 bp, 61.12% A\u0026thinsp;+\u0026thinsp;T) displaying structural homogeneity. Among ATP synthase genes, Atp8 presented the highest A\u0026thinsp;+\u0026thinsp;T content (69.15%), while CytB retained a moderate value (58.42%). The analysis of nucleotide diversity (π) across the genomic sequence revealed distinct variation patterns at different loci. Notable diversity peaks were observed, with π values approaching 0.004 in the regions associated with the ND1, ND4, and ND5 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of protein-coding genes in the mitochondrial genomes of Peruvian Creole sheep. The values presented correspond to the five mitochondrial genomes of PCS (B1\u0026ndash;B5), which present identical characteristics in gene length and A\u0026thinsp;+\u0026thinsp;T content.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene Length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;T Content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein Length (aa)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e319\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e327\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCox1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e515\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCox2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtp8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e69.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtp6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e226\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCox3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd4L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e459\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e607\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNd6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e380\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe phylogenetic analysis based on mitochondrial sequences revealed the genetic structure of different populations of \u003cem\u003eOvis aries\u003c/em\u003e and their close relatives. The phylogenetic tree, constructed using the maximum likelihood method, showed three main clades with high bootstrap support in most branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Representative sequences from various breeds and lineages of \u003cem\u003eO. aries\u003c/em\u003e from different regions of the world were included, as well as related species such as \u003cem\u003eO. ammon\u003c/em\u003e and \u003cem\u003eO. orientalis\u003c/em\u003e, which are grouped into well-defined clades.\u003c/p\u003e \u003cp\u003eThe mitochondrial genomes of interest, corresponding to Peruvian samples (B1, B2, B3, B4, and B5), belonging to Criollo sheep, clustered within Clade III, specifically in Subclade III, along with sheep breeds from Russia, New Zealand, and Brazil. However, these Peruvian samples do not form a monophyletic clade but are dispersed within the subclade, suggesting that they may have distinct mitochondrial origins.\u003c/p\u003e \u003cp\u003eSamples B1, B2, B3, and B4 formed a well-defined group with 90% BS support and exhibited a closer phylogenetic relationship with Criollo sheep sequences from Brazil, indicating a possible historical connection between these lineages. In contrast, sample B5 clusters more closely with a Yemule sheep from China. Clade I includes the wild lineages of \u003cem\u003eO. ammon\u003c/em\u003e and \u003cem\u003eO. orientalis\u003c/em\u003e, including the Argali and Asiatic Mouflon variants. In contrast, Clade II consists predominantly of domestic sheep breeds from China, such as the Duoma, Guide, and Qiaoke breeds. Clade III exhibited greater geographical diversity, with sheep breeds from Russia, New Zealand, Brazil, and Peru.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe analysis of relative synonymous codon usage (RSCU) in the mitochondrial genome of the PCS suggests distinct codon preferences, particularly in amino acids such as Arginine (Arg), Serine (Ser), and Threonine (Thr), which exhibited a marked bias toward specific codons (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;4). This pattern appears to align with previous studies indicating that codon usage bias (CUB) may be shaped by a combination of mutational pressure, natural selection, and genetic drift, with selection potentially acting on translation efficiency and accuracy in mitochondrial genes. The preference for certain synonymous codons has been documented across various taxa, including fishes, birds, and mammals, where overrepresented codons vary by species [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, given the high mutation rate characteristic of mitochondrial genomes, it is plausible that mutational pressure plays a predominant role in shaping codon preferences in \u003cem\u003eOvis aries\u003c/em\u003e, rather than selection for translational efficiency [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNucleotide diversity (π) is a measure of genetic variation within a population, indicating the average differences between pairs of DNA sequences. Certain genes exhibit higher nucleotide diversity in mitochondrial genomes, reflecting their evolutionary dynamics. Studies have identified significant variability in genes such as ND1, ND2, ND4, and ND5, which are part of the mitochondrial respiratory chain complex I. For instance, previous research reported multiple nucleotide substitutions in the ND1 gene, including both synonymous and missense mutations, highlighting its polymorphic nature. Similarly, the ND2, ND4, and ND5 genes have been observed to harbor numerous mutations, contributing to the overall genetic diversity of mitochondrial DNA [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These variations can influence mitochondrial function and have been associated with various diseases. The observed peaks in nucleotide diversity within the ND1, ND4, and ND5 regions suggest that these loci are subject to higher mutation rates or selective pressures, underscoring their importance in studies of mitochondrial genetics and evolution [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mitochondrial diversity observed in Peruvian Creole sheep reflects a complex pattern of genetic structuring, characterized by the absence of a monophyletic clade, suggesting multiple introduction events and gene flow with other ovine populations. This phenomenon is consistent with reports on African sheep breeds, where high genetic variability and the lack of clear phylogeographic structuring have been attributed to the historical mobility of flocks and transcontinental trade [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Likewise, the phylogenetic relationship between Peruvian and Brazilian sheep samples supports the hypothesis of a shared origin in South America, while the proximity of sample B5 to a Chinese sheep suggests the possibility of migration events or independent introductions. This pattern of genetic diversity is similar to that observed in Mediterranean sheep breeds, where genetic differentiation has been shaped by ancient migration routes and commercial interactions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The phylogeny of Peruvian Creole sheep, lacking a defined monophyletic grouping, indicates the presence of historical introgression, which may have contributed to the current genetic heterogeneity of these populations.\u003c/p\u003e \u003cp\u003eAdditionally, the phylogenetic analysis suggests that the genetic diversity in Peruvian Creole sheep may have been influenced by dispersal events like those observed in Central Asian sheep populations. A recent study on ancient sheep mitogenomes from China and Uzbekistan revealed that certain sheep populations acted as centers of genetic diffusion to new regions, facilitating adaptation to diverse environments [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The clustering of Peruvian samples within the same clade without a clear monophyletic structure reinforces the hypothesis that these lineages have undergone hybridization processes with different ovine populations over time. This phylogenetic behavior is consistent with that observed in Mediterranean and Bulgarian breeds, where genetic differentiation has been influenced by multiple dispersal and selection events [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, the population structure present in Peruvian Creole sheep not only reflects a history of multiple introductions but also a possible adaptive convergence resulting from natural and artificial selection in different environments.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe genetic diversity observed in Peruvian Creole sheep, characterized by the absence of a monophyletic clade, suggests multiple introduction events and historical gene flow with various ovine populations. This pattern aligns with findings in other geographically diverse breeds, where ancient migration routes, trade interactions, and human-driven selection have shaped genetic differentiation. The clustering of Peruvian samples within the same clade without a defined monophyletic structure indicates that these lineages have undergone hybridization with different populations over time. This underscores the importance of considering not only maternal lineages but also historical and anthropogenic factors when studying genetic diversity and conservation strategies. Further research, incorporating larger datasets and complementary genomic approaches, is essential to fully elucidate the evolutionary history and adaptive mechanisms of these populations, ensuring the preservation of their genetic resources.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is available in the NCBI repository (BioProject: XXXX).\u003c/p\u003e\n\u003cp\u003eReceived: ; Accepted:\u003c/p\u003e\n\u003cp\u003ePublished online:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our special appreciation and thanks to the owners of the PCS individuals located in district of Huayllay (Pasco, Peru) who kindly provided samples of their individuals \u0026nbsp;for this work. We also thank the Comunidad Campesina de Huayllay for supporting the logistic activities for field work. Finally, C.I.A. thanks Vicerrectorado de Investigación of UNTRM, and the Bioinformatics High-Performance Computing server of Universidad Nacional Agraria la Molina for providing resources for data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C. and C.I.A. conceived the study; F.-A.C., T.B.Y.-H. and C.I.A. planned and designed the field work; F.-A.C., T.B.Y.-H. D.F. and C.I.A. managed the animals and organized the collection of the samples; F.-A.C., T.B.Y.-H. and D.F. performed the experiments; R.E., Y.R., P.C., R.H.-Q. \u0026nbsp;and D.F. performed bioinformatic analysis; J.C. and C.I.A. were responsible of funding acquisition; F.-A.C., T.B.Y.-H., J.C. and C.I.A. were in charge of project administration. R.E., Y.R. and D.F. wrote the original draft; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the following two research projects: “Creación del servicio de agricultura de precisión en los departamentos de Lambayeque, Huancavelica, Ucayali y San Martín 4 departamentos-AGPRES” and “Mejoramiento de los servicios de investigación y transferencia de tecnologías para el manejo sostenible de la ganadería caprina en bosque seco y costa central en los departamentos de Tumbes, Piura, Lambayeque, Amazonas, La Libertad, Áncash y Lima-PROCAP” of the Ministry of Agrarian Development and Irrigation (MIDAGRI) of the Peruvian Government, with grant numbers CUI 2449640 and CUI \u0026nbsp;2506684, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample collection from the cattle specimen was conducted in accordance with the Peruvian National Law No. 30407: “Animal Protection and Welfare”.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDaly, K. 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This study analyzes the complete mitochondrial genome of five Peruvian Creole sheep (Ovis aries) individuals to assess their genetic variability and phylogenetic relationships. The mitochondrial genome exhibits a length of 16,617 base pairs (bp) with a guanine-cytosine (GC) content of 39%, encoding 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. Codon usage bias analysis reveals a marked preference for specific codons, particularly in Arginine (Arg), Serine (Ser), and Threonine (Thr), suggesting the influence of mutational pressure on mitochondrial gene evolution. Phylogenetic analysis places the PCS individuals within Clade III, alongside sheep breeds from Russia, Brazil, and New Zealand, but without forming a monophyletic group. The absence of monophyly suggests multiple historical introduction events and potential gene flow with different ovine populations, paralleling patterns observed in Mediterranean and African sheep breeds. The phylogenetic relationship of Peruvian Creole sheep with Brazilian and Chinese lineages highlights the complexity of their genetic background, indicating potential admixture and historical introgression. These findings underscore the need for further genomic research to clarify Peruvian Creole sheep's evolutionary history and support conservation efforts of their zoogenetic resources.","manuscriptTitle":"Comparative mitochondrial genomics of the Peruvian Creole sheep (Ovis aries)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 03:29:55","doi":"10.21203/rs.3.rs-6323309/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6290c630-7b89-4d39-903d-cb67fa248c11","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46700224,"name":"Biological sciences/Genetics/Evolutionary biology"},{"id":46700225,"name":"Biological sciences/Genetics/Genomics"},{"id":46700226,"name":"Biological sciences/Genetics/Animal breeding"}],"tags":[],"updatedAt":"2025-09-03T13:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-02 03:29:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6323309","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6323309","identity":"rs-6323309","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}