{"paper_id":"aaa359e9-622e-486d-9ee4-aaffdb8331d0","body_text":"Mitochondrial and Retroviral Markers Reveal High Genetic Diversity and Regional Structure in the Lebanese Awassi Sheep | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial and Retroviral Markers Reveal High Genetic Diversity and Regional Structure in the Lebanese Awassi Sheep Jeanne El Hage, Frédéric Boyer, Barbara Viginier, Christophe Terzian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8807670/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The Awassi is a native sheep breed of Lebanon with high historical and economic value. Despite its importance, its genetic background has not been characterized, leaving limited understanding of its origins, diversity, and regional connections. This study aimed to characterize the genetic diversity and population structure of Awassi sheep from Lebanon and neighboring Syria using two complementary genetic systems that trace distinct evolutionary histories: maternal mitochondrial DNA and insertional polymorphisms of endogenous Jaagsiekte sheep retroviruses. Genetic analyses were conducted on two hundred seventy-seven animals. Sequencing of a one thousand thirty-four base pair fragment of the mitochondrial cytochrome b gene identified eighty-six maternal haplotypes that clustered into four haplogroups (A, B, C, and E). Maternal haplotype diversity was high, and most individuals belonged to haplogroups A and B, indicating contributions from both western and eastern maternal lineages. Retroviral insertion profiling showed that the retroviral type carrying only endogenous Jaagsiekte sheep retrovirus element eighteen was predominant. A Mediterranean retroviral type carrying both endogenous Jaagsiekte sheep retrovirus eighteen and seven was also present, especially in coastal flocks, suggesting ancient gene flow through maritime trade. Other retroviral combinations were rare. Analysis of endogenous Jaagsiekte sheep retrovirus six revealed a high frequency of solo long terminal repeat alleles, indicating an ancient recombination event. Approximately half of the animals were heterozygous for this allele and some homozygous. No pre-integration alleles were detected, confirming fixation of endogenous Jaagsiekte sheep retrovirus six in domestic sheep. Lebanese Awassi sheep exhibit substantial genetic diversity across both maternal and retroviral systems, reflecting a complex population history shaped by multiple lineages and regional exchanges. This study provides the first comprehensive genetic characterization of this native breed and contributes to broader understanding of sheep domestication in the Middle East, while supporting evidence-based conservation of this valuable genetic resource. Biological sciences/Evolution Biological sciences/Genetics Awassi sheep genetic diversity mitochondrial DNA endogenous Jaagsiekte sheep retrovirus population structure Middle East domestication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION The Awassi is one of the oldest and most widespread sheep breeds in the Arabian Peninsula, with historical records dating back to at least the 17th century BCE. It is distributed across Lebanon, Syria, Jordan, Palestine, Iraq, and Saudi Arabia, and plays a crucial socio-economic role in these regions. Lebanon, situated in the Levant, lies at a crossroads of historical animal trade routes (Phoenician, Roman, Silk Road, etc.) and was near the cradle of sheep domestication in the early Neolithic (Meadows et al. 2007 ). Despite the breed’s importance and antiquity, its genetic makeup has not been formally studied. Locally adapted livestock conservation is now recognized as vital (FAO and others), but no genetic baseline exists for Lebanese Awassi. Domestic sheep ( Ovis aries ) have been analyzed with various genetic markers: mitochondrial DNA (mtDNA) to trace maternal lineages (e.g. Bruford et al. 2003 ; Meadows et al. 2007 ), Y-chromosome markers for paternal lineages, microsatellites, endogenous retroviral insertions, and most recently genome-wide SNPs (Kijas et al. 2012 ; Alberto et al. 2018 ; Cumer et al. 2021 ). Mitochondrial genes, like cytochrome b (CytB), are widely used for maternal phylogeography and haplogroup assignment. Meadows et al. ( 2011 ) showed that CytB haplotypes reliably reflect major sheep mitochondrial lineages (haplogroups A–E) identified by whole mitogenomes. Endogenous Jaagsiekte sheep retroviruses (enJSRVs) are another powerful marker: their insertional presence/absence patterns distinguish ancient “primitive” breeds from later-derived modern breeds (Chessa et al. 2009 ). A particular focus is the LTR structure of enJSRV-6, a provirus present in all sheep genomes; recombination between its LTRs can produce a solo LTR, a marker of ancient integration (Arnaud et al., 2007 ; Perucatti et al., 2022 ; Verneret et al., 2024 ). Here we present the first genetic survey of Lebanese Awassi sheep using these two markers. We sequenced the mitochondrial CytB gene and genotyped seven enJSRV loci (6, 7, 8, 15, 16, 18, 22) in 277 sheep from Lebanon (and two Syrian flocks) to assess maternal diversity, haplogroup composition, and retroviral insertion patterns. This combined approach aims to reveal the maternal origins and demographic history of the breed, its relationship to regional sheep diversity, and to identify retroviral markers of evolutionary significance. RESULTS Genetic characterization based on mtDNA The Cyt-b fragments (1034 bp) were sequenced for 277 Lebanese and Syrian Awassi. The average nucleotide composition of these sequences was: T (27.0%), C (29.0%), A (30.7%) and G (13.3%). As shown in Table 1 , these individuals correspond to 86 different haplotypes with a haplotype diversity of Hd = 0.9013. Next, to evaluate the relationship within and between haplotypes, the cytB sequences data set was used with the reference sequences in the construction of a median joining network (MJ) (Fig. 2A). The MJ network revealed that the Awassi sheep is separated into four distinct maternal lineages called A, B, C and E. The majority of animals grouped into two clades A (n = 100) and B (n = 115) which represents ≈ 77% of the animals tested. The geographical distribution of the haplogroups is shown in Fig. 2B. Table 1 Mitochondrial haplogroups phylogenetic variation of the Awassi sheep Haplogroups A B C E Individuals 100 115 50 12 Variables Sites (S) 42 39 14 5 Nucleotide diversity (π) 1,2 x 10 − 3 1,17 x 10 − 3 1,13 x 10 − 3 1,16 x 10 − 3 Haplotypes 33 36 12 5 Haplotype Diversity (Hd) 0.686 ± 0.00293 0,715 ± 0,00226 0,728 ± 0.00313 0.758 ± 0.00859 The average number of nucleotide differences (Kxy) and sequence diversity (Dxy) between haplogroups were calculated to confirm the different lineages. Haplogroups HA and HB were separated by an average of 3.182 substitutions, for HB–HE (Kxy = 9.304) and HB–HC (Kxy = 12.7) (Table 2 ). Comparison of sequence diversity between the same lineages as above showed the same trend ranging from a low of 0.48% for HA–HB to the highest variation of 1.228% for HB–HC. Table 2 Nucleotide differences (Kxy) and genetic diversity (Dxy) among four clades of domestic sheep HA HB HC Dxy % Kxy Dxy % Kxy Dxy % Kxy H B 0.308 3.182 H C 1.041 10.763 1.228 12.701 H E 0.713 7.374 0.900 9.304 0.473 4.890 Among the Awassi population of Lebanon, two major haplogroups HA and HB formed a star-like tree with one central haplotype (Hap_2 and Hap_36, respectively) which is consistent with population expansions. Genetic characterization using enJSRVs All 277 DNA samples were screened by locus-specific PCR for six insertionally polymorphic enJSRV loci (enJSRV-7, -8, -15, -16, -18 and − 22) as described in Materials and Methods. For loci in which a provirus was present, we obtained matching 5′ and 3′ proviral amplicons, consistent with full-length proviral insertion. In addition, an “empty-site” amplicon (pre-integration size) was amplified in many samples and, where ambiguous, its identity was confirmed by Sanger sequencing; the concurrent recovery of provirus and empty-site products in individual PCRs indicates heterozygosity at the corresponding integration loci in those animals (see representative gel in Fig. 3). Density maps were produced from the 254 Lebanese samples to visualize the frequency and spatial distribution of each enJSRV. enJSRV-18 was fixed in our Lebanese sample set (detected in 100% of animals), whereas enJSRV-7 was present in 38 animals (≈ 15%; Fig. 5A–B). The remaining loci occurred at lower frequencies: enJSRV-15 (n = 17, 6.7%), enJSRV-16 (n = 27, 10.6%) and enJSRV-22 (n = 14, 5.5%) (Figure. 5C–E). enJSRV-8 was not detected in any Lebanese sample. Geographically, enJSRV-7 was more frequent along western and southern coastal localities, enJSRV-15 showed a relative concentration in the north-west, and enJSRV-16 exhibited a predominantly southern distribution (Figure. 4). These spatial patterns suggest differential historical and/or contemporary gene flow across the Lebanese landscape. Retrotype distribution in Awassi sheep In total, 277 animals were analyzed for enJSRV retrotypes (254 Lebanese samples and 23 from two Syrian herds). Retrotype classification was based on the presence/absence profile of insertionally polymorphic enJSRVs following Chessa et al. ( 2009 ). Across Lebanese flocks the R2 retrotype defined by the presence of enJSRV-18 only and considered a marker of “modern” breeds, was the most frequent, occurring in 68.1% of Lebanese animals (Fig. 5A). The R4 retrotype (enJSRV-18 + enJSRV-7), commonly associated with Mediterranean populations, was the second most common retrotype in Lebanon (overall 14.6%). However, R4 showed marked spatial heterogeneity, varying from 0% to ~ 50% among herds (Fig. 5B). R4 was significantly less frequent in north-eastern Lebanon (Baalbek, Hermel, Beqaa) and significantly more frequent and more evenly distributed among coastal and some south-eastern localities (including Rachaiya) on the Mediterranean side (Kruskal–Wallis test, p = 0.005377, < 0.01). The two sampled Syrian herds exhibited a substantially higher R4 prevalence (> 50%), a difference that is highly significant when compared with Lebanese flocks (Kruskal–Wallis test, p = 0.000164). Beyond R2 and R4, several other retrotypes were detected at lower frequencies. The RB retrotype (enJSRV-18 + enJSRV-16) was the most frequent after R2/R4, with an overall prevalence of 10.6% in Lebanese Awassi. RB was not detected in Baalbek but reached 13.3% in Saida, 8.3% in Hermel, and 7.0% in the Beqaa; the highest RB frequency was observed in the Syrian Al-Qusayr herd (66.7%). The RA retrotype (enJSRV-18 + enJSRV-15) occurred in 6.3% of Lebanese animals (Supplementary Figure S1 ). The R6 retrotype (enJSRV-18 + enJSRV-22), which is more typical of northern European and some Caucasus populations, was comparatively rare in Lebanon (5.5%; 14 samples), and these R6 carriers were scattered across 14 different herds (Supplementary Figure S2). Retrotypes defined by combinations of three or more insertions were uncommon: for example, RF (18 + 7+16) was observed in ~ 1.6% of animals, while RD (18 + 15+16) was detected in ~ 0.4%. The four-locus combination RG (18 + 7+16 + 22) and RH (18 + 16+22) were each found in a single individual. At the herd level, retrotype richness was highest in the Kobayat commercial flock (northern Lebanon) and in the two Syrian flocks, each displaying at least five distinct retrotypes. This pattern is consistent with greater animal movement and commercial exchange in these herds and highlights how management practices influence local retrotype diversity. Overall, the retrotype distribution in Lebanese Awassi reflects a predominance of modern-type ancestry (R2) combined with measurable Mediterranean and regionally specific signatures (R4, RB, RA), shaped by both historical dispersal routes and contemporary flock management. enJSRV-6 solo-LTR in Lebanese Awassi sheep enJSRV-6, a deeply conserved endogenous retrovirus previously reported to be fixed across Ovis genomes (Arnaud et al., 2007 ), was initially included here as an internal control for enJSRV genotyping. PCR assays targeting the 5′ and 3′ proviral ends, together with the empty-site PCR, produced an unexpected pattern in a subset of samples. Fifteen of the 277 tested animals yielded clear 5′ and 3′ LTR amplicons but lacked the corresponding 5′/3′ provirus amplicons (Fig. 6). In several additional samples the empty-site assay amplified a fragment of ≈ 750 bp rather than the expected ≈ 302 bp; Sanger sequencing of representative products confirmed that these anomalous bands correspond to solo long terminal repeat (solo-LTR) alleles derived from recombination between proviral LTRs (Mager & Stoye, 2015 ; Perucatti et al., 2022 ; Verneret et al., 2024 ). Taken together, the PCR and sequencing results indicate three genotypic classes at the enJSRV-6 locus: (i) homozygous for the full-length provirus (hereafter “Homo/Prov”); (ii) heterozygous, carrying one provirus allele and one solo-LTR allele (“Hetero/Prov-LTR”); and (iii) homozygous for the solo-LTR (“Homo/LTR”) (Fig. 7). Density maps constructed from the 254 Lebanese samples (Syrian samples treated separately) show that the three genotypes are widespread: 47.64% Homo/Prov, 47.64% Hetero/Prov-LTR and 4.72% Homo/LTR (Fig. 7; Table 3 ). Table 3 Table summarizing the PCR results of enJSRV-6 in terms of zygosity for all the samples tested enJSRV-6 Homo/Pro Hetero/Pro-LTR Homo/LTR Provirus Positive Positive Negative Solo LTR Positive Positive Positive Empty Locus Negative Positive Positive Number of samples 126 136 15 The geographic distribution of genotypes is non-random. The Hetero/Prov-LTR genotype is significantly enriched in southwestern Lebanon (Saida, Jezzine, Nabatieh) and is broadly distributed among flocks in that region (Kruskal–Wallis test, p = 0.000578; Fig. 8A). Correspondingly, the combined frequency of animals carrying at least one solo-LTR allele (Hetero/Prov-LTR + Homo/LTR) is higher in these southern localities than elsewhere (Kruskal–Wallis test, p = 0.042320). Two herds in Nabatieh (total n = 10) are extreme examples: none of the sampled individuals in these herds were Homo/Prov; all were either Hetero/Prov-LTR or Homo/LTR (Fig. 8B). These findings indicate that (i) enJSRV-6 is present in all sampled genomes, consistent with an ancient integration event, and (ii) homologous recombination between LTRs has produced a frequent solo-LTR allele that has achieved substantial prevalence in several Lebanese flocks. The high frequency and partial homozygosity of the solo-LTR allele suggests that the recombination event is not recent and has been propagated through multiple generations. Geographic clustering of the solo-LTR allele may reflect historical founder effects, restricted gene flow, or local breeding practices; elucidating the relative contribution of these processes will require broader regional sampling and complementary autosomal data. DISCUSSION Mitochondrial DNA analysis provides a maternal perspective on the evolutionary history and demographic composition of livestock populations. In the Lebanese Awassi, Cyt-b sequence data revealed a high level of maternal diversity and the co-occurrence of four established sheep haplogroups: A (≈ 36%), B (≈ 41.5%), C (≈ 18%) and the rare E (≈ 4.5%). Haplogroups A, B and C are widely distributed and collectively represent the major maternal lineages in domestic sheep (Bruford et al. 2003 ; Meadows et al. 2007 ). The near-codominance of A and B in Lebanon contrasts with some “improved” Awassi populations, which show a stronger bias toward haplogroup B (Meadows et al. 2007 ) and indicates that the Lebanese Awassi herd has retained substantial ancestral maternal variation. The presence of haplogroup C at appreciable frequency, and the detection of the relatively uncommon haplogroup E (often associated with fat-tailed Middle Eastern breeds), further point to multiple maternal inputs into the Lebanese population. Collectively, these results are consistent with Lebanon’s historical role as a crossroads of animal movement and trade during and after the Neolithic, and with a history of repeated admixture rather than a single, strongly bottlenecked maternal origin. The enJSRV insertional polymorphisms provide a complementary, largely independent signal of sheep demographic history. In our sample enJSRV-18 was ubiquitous, and the R2 retrotype (enJSRV-18 alone) predominated (≈ 68%), consistent with a substantial contribution from lineages associated with the second wave of sheep dispersal that produced many modern breeds (Chessa et al. 2009 ). The Mediterranean R4 retrotype (enJSRV-18 + enJSRV-7) was also present (≈ 14.6%) and exhibited a clear geographic bias: it was more frequent in coastal and western localities (including historically important Phoenician ports) and comparatively rare in eastern highland regions (e.g. Baalbek, Hermel, Beqaa). This spatial pattern suggests that maritime and coastal trade routes have left a detectable genetic imprint on local flocks, whereas Lebanon’s central mountain ranges have acted as partial barriers to gene flow, producing contrasting retrotype compositions between coastal and inland flocks. The low but notable frequency of R6 (≈ 5.5%), a type more typical of northern Europe and some Caucasus populations (Chessa et al. 2009 ; Schroeder et al. 2017 ), indicates limited introgression from more distant sources, plausibly driven by commercial exchanges rather than historical dispersals. The Kobayat herd, a commercial flock with frequent livestock exchanges, displayed the highest retrotype diversity, which underlines the sensitivity of enJSRV profiles to recent management practices and animal movement. Overall, the combined mtDNA and enJSRV data portray a population shaped by both ancient demographic processes and ongoing anthropogenic-mediated gene flow. A particularly noteworthy observation is the high prevalence of a solo-LTR allele at the enJSRV-6 locus: more than half of the tested animals carried a solo-LTR in at least one chromosomal copy (54.5% of samples), and a minority were homozygous for the solo-LTR. Solo-LTRs arise by homologous recombination between 5′ and 3′ LTRs of a provirus and accumulate over long evolutionary timescales (Mager & Stoye 2015 , Perucatti et al., 2022 ; Verneret et al., 2024 ). The absence of empty pre-integration alleles for enJSRV-6 and the presence of solo-LTRs are consistent with an ancient insertion that predates the Capra–Ovis split and has since undergone recombination in certain lineages (Arnaud et al. 2007 ). The substantial frequency and partial homozygosity observed here indicate that the recombination event is not recent and has been transmitted through multiple generations. Because enJSRV-6 has been thought to be fixed across sheep, the detection of a frequent solo-LTR allele in Lebanese Awassi (and reported previously in some Turkish local breeds) suggests regional or lineage-specific evolutionary dynamics (Chessa et al., 2009 ; Ayanoğlu, 2013 ; Demirci et al., 2013 ). The geographic heterogeneity in solo-LTR frequency (e.g. enrichment in southern localities) could reflect historical founder effects, drift in relatively closed breeding systems, or selection at linked loci. Regardless of mechanism, the enJSRV-6 solo-LTR appears to be a stable genomic marker that may be useful for reconstructing deep phylogeographic relationships among Middle Eastern sheep populations, provided its distribution is assessed in broader comparative samples. The joint evidence from mtDNA and enJSRV retrotypes supports a scenario in which Lebanese Awassi sheep are the product of layered demographic events: an ancient substrate of regionally adapted stock, later admixture with populations carrying “modern” retrotypes, and ongoing local exchanges that introduce additional diversity. This mosaic is consistent with models of multiple dispersal waves during sheep domestication and subsequent historical translocations across the Mediterranean and Near East (Chessa et al. 2009 ; Meadows et al. 2011 ). From a conservation and management perspective, the high mitochondrial and retroviral diversity documented here is an asset. It argues for conservation strategies that preserve within-breed variability by avoiding indiscriminate crossbreeding and by maintaining representative herds from different geographic regions. Herds that retain uncommon mitolineages or rare retrotypes (and those harboring distinctive enJSRV-6 solo-LTR frequencies) merit targeted conservation attention and could serve as repositories for regional genetic variation. Several limitations temper the inferences that can be drawn from the present study. Mitochondrial markers reflect only the maternal lineage and do not capture the autosomal or paternal genetic variation that shapes most phenotypic traits; likewise, enJSRV insertions provide a useful but partial record of historical events. To obtain a fuller picture of population structure and admixture, genome-wide autosomal SNP data and Y-chromosome markers should be incorporated in future work. Broader geographic sampling across neighboring countries (Syria, Jordan, Turkey) would contextualize the Lebanese patterns and test hypotheses about routes of gene flow. Finally, formal demographic modelling (e.g. using coalescent or approximate Bayesian computation approaches) and temporal sampling -where feasible- would permit more precise reconstruction of the timing and magnitude of admixture and expansion events. This study establishes a baseline genetic portrait of the Lebanese Awassi sheep, demonstrating considerable maternal and retroviral diversity and identifying an intriguing regional signature in the enJSRV-6 solo-LTR. These results refine our understanding of sheep population history in the Levant and provide concrete targets for conservation and future genomic investigation. METHODS Sampling and DNA extraction Blood samples were collected from 254 presumably unrelated Awassi sheep in Lebanon (47 herds across 12 administrative departments) and from 23 sheep in two Syrian herds (Fig. 1). Sampling was performed by licensed veterinarians from the Lebanese Agricultural Research Institute (LARI), with informed consent obtained from animal owners. All procedures were conducted in strict accordance with internationally accepted animal welfare standards, following the principles of the ARRIVE guidelines and relevant national regulations for the care and use of animals in research, ensuring minimal stress and discomfort to the animals. Genomic DNA was extracted from EDTA-anticoagulated whole blood using the GenElute™ Blood Genomic DNA kit (Sigma-Aldrich), following the manufacturer’s instructions. DNA concentration and purity were assessed by spectrophotometry. The sampling protocol and experimental procedures were reviewed and approved by the Research Ethics Committee (REC) of the Higher Center for Research (HCR) at the Holy Spirit University of Kaslik (USEK). Mitochondrial CytB DNA PCR and sequencing A 1272-bp fragment of mitochondrial CytB (positions 14,078–15,349 of Ovis aries reference genome AF010406) was amplified using primers cytbF and cytbR (Meadows et al. 2005 ). PCR reactions were 50 µL volume, containing: 1× Taq buffer (with 1.5 mM MgCl₂), 200 µM of each dNTP, 0.5 µM of each primer, and 1 U Taq DNA Polymerase (Qiagen Taq PCR Core Kit). Cycling conditions were: initial denaturation at 95°C for 5 min; 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were verified by agarose gel electrophoresis (1% agarose, ethidium bromide) and purified with the GenElute™ PCR Clean-up Kit (Sigma-Aldrich). Cycle sequencing of both strands was performed using the BigDye® Terminator v1.1 kit (Applied Biosystems) under standard conditions. Labeled products were run on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequences were assembled and edited in ChromasPro v.1.7.5.4 (Technelysium). Ambiguous bases at read ends were trimmed, yielding a final alignment of 1034 bp per sample. For the data analysis, indices of sequence variation, including the total number of polymorphic sites, nucleotide diversity (π), mean number of nucleotide differences (D), and average number of nucleotide substitutions per site (K), were calculated using DnaSP v6.0.73 (Rozas et al., 2017 ). Haplotype structure and diversity parameters were also estimated using the same software. A median-joining (MJ) network of the mitochondrial cytochrome b (Cyt-b) region was constructed with NETWORK v4 (Bandelt et al., 1999 ) to infer relationships among Awassi haplotypes. The network was built from the Cyt-b sequences generated in this study together with ten domestic reference sequences representing the five major mitochondrial haplogroups previously described in sheep (Meadows et al., 2011 ): Haplogroup A (HM236174, HM236175), Haplogroup B (HM236176, HM236177), Haplogroup C (HM236178, HM236179), Haplogroup D (HM236180, HM236181), and Haplogroup E (HM236182, HM236183). The resulting network allowed visualization of mutational steps and genealogical relationships among Lebanese Awassi haplotypes and their connection to global Ovis aries mitochondrial lineages. DNA amplification of enJSRV The extracted genomic DNA was also used as a template for the detection of endogenous retroviruses. The presence of seven enJSRV-6, -7, -8, -15, -16 and − 18 and − 22 was evaluated by PCR as already described by (Arnaud et al. 2007 ). The amplification of each enJSRV provirus was obtained by two sets of PCR reactions (5’- and 3’-PCR). The presence of solo LTRs was also assessed, for each provirus, using three distinct PCR assays specifically designed to amplify solo LTRs and the empty genomic DNA around the proviral integration site. All the primers used in this study are presented in supplementary fig. S1 . Among the seven enJSRVs, enJRSV-6 is the oldest and used as a positive control of enJSRV amplification while the six other enJSRVs are insertionally polymorphic within the Ovis aries genomes (Arnaud et al. 2007 ). The reactions were carried out in a final volume of 50µl, containing 5µl of DNA template, 1x Taq Buffer (Tris-Cl, KCl, (NH 4 ) 2 SO 4 , 1.5 mM MgCl 2 ; pH 8.7), 1x Q solution, 200µM of each dNTP, 2.5U of HotStar Taq ADN Polymerase (« HotStar Taq® DNA Polymerase », Qiagen, Germany) et 0.2µM of each primer. Retrotypes for each animal were assigned based on the presence/absence profile of enJSRV insertions following Chessa et al. ( 2009 ): R2 (only enJSRV-18 present), R4 (enJSRV-18 and − 7), R6 (enJSRV-18 and − 22), R11 (enJSRV-18 and − 8), as well as two novel categories defined here: RA (enJSRV-18 + -15) and RB (enJSRV-18 + -16). Other combinations involving three or more proviruses were noted but were rare. Declarations Ethics approval and consent to participate This study involved non-experimental field sampling of client-owned animals. Blood collection was performed by licensed veterinarians from the Lebanese Agricultural Research Institute (LARI) in compliance with international guidelines for the ethical treatment of animals, including the International Council for Laboratory Animal Science (ICLAS) ethical principles for animal research. Sampling procedures did not involve anesthesia, euthanasia, or experimental interventions. In accordance with GSE policies for studies involving client-owned animals, informed consent was obtained from all animal owners prior to sampling, and all veterinary procedures adhered to recognized best practices. Under Lebanese regulations (non-experimental veterinary sampling of domestic livestock), this work did not require formal approval from an animal ethics committee or institutional review board; accordingly, no approval number is issued. Consent for publication Not applicable Data availability The mitochondrial cytochrome b sequences generated in this study have been deposited in the GenBank nucleotide database (INSDC) under accession numbers PX994374–PX994861 . Public release of the records will be on 18 Feb, 2026. Additional raw data supporting the findings of this study, including detailed genotyping matrices and supplementary metadata, are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was cofounded by the Lebanese Agricultural Research Institute (LARI) and Ecole Pratique des Hautes Etudes (EPHE) Authors’ contributions Conceptualization: Christophe Terzian, Alain Abi-Rizk Project administration: Frederic Arnaud Investigation: Jeanne El Hage Formal analysis & methodology: Jeanne El Hage, Frédérick Arnaud, Frédéric Boyer, Barbara Viginier, François Pompanon, Data interpretation: Jeanne El Hage, Frédérick Arnaud, Frédéric Boyer Supervision: Frédérick Arnaud, Alain Abi-Rizk Writing – review & editing: All authors Final approval of manuscript: All authors References Alberto, F. J. et al. Convergent genomic signatures of domestication in sheep and goats. Nat. Commun. 9 (1), 813. https://doi.org/10.1038/s41467-018-03206-y (2018). Arnaud, F. et al. A paradigm for virus–host coevolution: Sequential counter-adaptations between endogenous and exogenous retroviruses. 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Animals 12 (20), 2834. https://doi.org/10.3390/ani12202834 (2022). Pereira, F. et al. Genetic signatures of a Mediterranean influence in Iberian Peninsula sheep husbandry. Mol. Biol. Evol. 23 (7), 1420–1426. https://doi.org/10.1093/molbev/msl006 (2006). Peter, C. et al. Genetic diversity and subdivision of 57 European and Middle-Eastern sheep breeds. Anim. Genet. 38 (1), 37–44. https://doi.org/10.1111/j.1365-2052.2006.01561.x (2007). Rozas, J. et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 34 (12), 3299–3302. https://doi.org/10.1093/molbev/msx248 (2017). Schroeder, O. et al. Endogenous retroviral insertions indicate a secondary introduction of domestic sheep lineages to the Caucasus and Central Asia between the Bronze and Iron Age. Genes 8 (6), 154. https://doi.org/10.3390/genes8060154 (2017). Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123 (3), 585–595. https://doi.org/10.1093/genetics/123.3.585 (1989). Tapio, M. et al. Sheep mitochondrial DNA variation in European, Caucasian, and Central Asian areas. Mol. Biol. Evol. 23 (9), 1776–1783. https://doi.org/10.1093/molbev/msl043 (2006). Uzun, M. et al. Genetic relationships among Turkish sheep. Genet. Selection Evol. 38 (5), 513–524. https://doi.org/10.1186/1297-9686-38-5-513 (2006). Verneret, M., Lannes, B., Arnaud, F. & Palmarini, M. A genome-wide study of ruminants uncovers two endogenous retrovirus families with recent activity. Mobile DNA (2024). (Advance online publication). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pptx Annex1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 15 May, 2026 Reviewers agreed at journal 15 May, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 02 Mar, 2026 Editor assigned by journal 02 Mar, 2026 Editor invited by journal 24 Feb, 2026 Submission checks completed at journal 18 Feb, 2026 First submitted to journal 18 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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15:34:42\",\"extension\":\"pptx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":6609744,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterial.pptx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8807670/v1/8f7972df5fd8f5278d276ede.pptx\"},{\"id\":104402305,\"identity\":\"dd16681a-2a18-4e5c-a8f2-acf481d9be29\",\"added_by\":\"auto\",\"created_at\":\"2026-03-11 12:15:00\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":20933,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Annex1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8807670/v1/cb62943a831a5b3c84f61f0b.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Mitochondrial and Retroviral Markers Reveal High Genetic Diversity and Regional Structure in the Lebanese Awassi Sheep\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eThe Awassi is one of the oldest and most widespread sheep breeds in the Arabian Peninsula, with historical records dating back to at least the 17th century BCE. It is distributed across Lebanon, Syria, Jordan, Palestine, Iraq, and Saudi Arabia, and plays a crucial socio-economic role in these regions. Lebanon, situated in the Levant, lies at a crossroads of historical animal trade routes (Phoenician, Roman, Silk Road, etc.) and was near the cradle of sheep domestication in the early Neolithic (Meadows et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). Despite the breed\\u0026rsquo;s importance and antiquity, its genetic makeup has not been formally studied. Locally adapted livestock conservation is now recognized as vital (FAO and others), but no genetic baseline exists for Lebanese Awassi.\\u003c/p\\u003e \\u003cp\\u003eDomestic sheep (\\u003cem\\u003eOvis aries\\u003c/em\\u003e) have been analyzed with various genetic markers: mitochondrial DNA (mtDNA) to trace maternal lineages (e.g. Bruford et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Meadows et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e), Y-chromosome markers for paternal lineages, microsatellites, endogenous retroviral insertions, and most recently genome-wide SNPs (Kijas et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Alberto et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Cumer et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Mitochondrial genes, like cytochrome b (CytB), are widely used for maternal phylogeography and haplogroup assignment. Meadows et al. (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e) showed that CytB haplotypes reliably reflect major sheep mitochondrial lineages (haplogroups A\\u0026ndash;E) identified by whole mitogenomes. Endogenous Jaagsiekte sheep retroviruses (enJSRVs) are another powerful marker: their insertional presence/absence patterns distinguish ancient \\u0026ldquo;primitive\\u0026rdquo; breeds from later-derived modern breeds (Chessa et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). A particular focus is the LTR structure of enJSRV-6, a provirus present in all sheep genomes; recombination between its LTRs can produce a solo LTR, a marker of ancient integration (Arnaud et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Perucatti et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Verneret et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eHere we present the first genetic survey of Lebanese Awassi sheep using these two markers. We sequenced the mitochondrial CytB gene and genotyped seven enJSRV loci (6, 7, 8, 15, 16, 18, 22) in 277 sheep from Lebanon (and two Syrian flocks) to assess maternal diversity, haplogroup composition, and retroviral insertion patterns. This combined approach aims to reveal the maternal origins and demographic history of the breed, its relationship to regional sheep diversity, and to identify retroviral markers of evolutionary significance.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGenetic characterization based on mtDNA\\u003c/h2\\u003e \\u003cp\\u003eThe Cyt-b fragments (1034 bp) were sequenced for 277 Lebanese and Syrian Awassi. The average nucleotide composition of these sequences was: T (27.0%), C (29.0%), A (30.7%) and G (13.3%). As shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, these individuals correspond to 86 different haplotypes with a haplotype diversity of Hd\\u0026thinsp;=\\u0026thinsp;0.9013. Next, to evaluate the relationship within and between haplotypes, the cytB sequences data set was used with the reference sequences in the construction of a median joining network (MJ) (Fig.\\u0026nbsp;2A). The MJ network revealed that the Awassi sheep is separated into four distinct maternal lineages called A, B, C and E. The majority of animals grouped into two clades A (n\\u0026thinsp;=\\u0026thinsp;100) and B (n\\u0026thinsp;=\\u0026thinsp;115) which represents\\u0026thinsp;\\u0026asymp;\\u0026thinsp;77% of the animals tested. The geographical distribution of the haplogroups is shown in Fig.\\u0026nbsp;2B.\\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 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eMitochondrial haplogroups phylogenetic variation of the Awassi sheep\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHaplogroups\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eB\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eC\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eE\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eIndividuals\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e100\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e115\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e12\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eVariables Sites (S)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e42\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e39\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e14\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eNucleotide diversity (π)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1,2 x 10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1,17 x 10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1,13 x 10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1,16 x 10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHaplotypes\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e33\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e36\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e12\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHaplotype Diversity (Hd)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.686\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.00293\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0,715\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0,00226\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0,728\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.00313\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.758\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.00859\\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\\u003eThe average number of nucleotide differences (Kxy) and sequence diversity (Dxy) between haplogroups were calculated to confirm the different lineages. Haplogroups HA and HB were separated by an average of 3.182 substitutions, for HB\\u0026ndash;HE (Kxy\\u0026thinsp;=\\u0026thinsp;9.304) and HB\\u0026ndash;HC (Kxy\\u0026thinsp;=\\u0026thinsp;12.7) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Comparison of sequence diversity between the same lineages as above showed the same trend ranging from a low of 0.48% for HA\\u0026ndash;HB to the highest variation of 1.228% for HB\\u0026ndash;HC.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eNucleotide differences (Kxy) and genetic diversity (Dxy) among four clades of domestic sheep\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\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 \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eHA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eHB\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c7\\\" namest=\\\"c6\\\"\\u003e \\u003cp\\u003eHC\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDxy %\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eKxy\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eDxy %\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eKxy\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eDxy %\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eKxy\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eH B\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.308\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.182\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eH C\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.041\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e10.763\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.228\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e12.701\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eH E\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.713\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.374\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.900\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e9.304\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.473\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e4.890\\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\\u003eAmong the Awassi population of Lebanon, two major haplogroups HA and HB formed a star-like tree with one central haplotype (Hap_2 and Hap_36, respectively) which is consistent with population expansions.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eGenetic characterization using enJSRVs\\u003c/h3\\u003e\\n\\u003cp\\u003eAll 277 DNA samples were screened by locus-specific PCR for six insertionally polymorphic enJSRV loci (enJSRV-7, -8, -15, -16, -18 and \\u0026minus;\\u0026thinsp;22) as described in Materials and Methods. For loci in which a provirus was present, we obtained matching 5\\u0026prime; and 3\\u0026prime; proviral amplicons, consistent with full-length proviral insertion. In addition, an \\u0026ldquo;empty-site\\u0026rdquo; amplicon (pre-integration size) was amplified in many samples and, where ambiguous, its identity was confirmed by Sanger sequencing; the concurrent recovery of provirus and empty-site products in individual PCRs indicates heterozygosity at the corresponding integration loci in those animals (see representative gel in Fig.\\u0026nbsp;3).\\u003c/p\\u003e \\u003cp\\u003eDensity maps were produced from the 254 Lebanese samples to visualize the frequency and spatial distribution of each enJSRV. enJSRV-18 was fixed in our Lebanese sample set (detected in 100% of animals), whereas enJSRV-7 was present in 38 animals (\\u0026asymp;\\u0026thinsp;15%; Fig.\\u0026nbsp;5A\\u0026ndash;B). The remaining loci occurred at lower frequencies: enJSRV-15 (n\\u0026thinsp;=\\u0026thinsp;17, 6.7%), enJSRV-16 (n\\u0026thinsp;=\\u0026thinsp;27, 10.6%) and enJSRV-22 (n\\u0026thinsp;=\\u0026thinsp;14, 5.5%) (Figure. 5C\\u0026ndash;E). enJSRV-8 was not detected in any Lebanese sample. Geographically, enJSRV-7 was more frequent along western and southern coastal localities, enJSRV-15 showed a relative concentration in the north-west, and enJSRV-16 exhibited a predominantly southern distribution (Figure. 4). These spatial patterns suggest differential historical and/or contemporary gene flow across the Lebanese landscape.\\u003c/p\\u003e\\n\\u003ch3\\u003eRetrotype distribution in Awassi sheep\\u003c/h3\\u003e\\n\\u003cp\\u003eIn total, 277 animals were analyzed for enJSRV retrotypes (254 Lebanese samples and 23 from two Syrian herds). Retrotype classification was based on the presence/absence profile of insertionally polymorphic enJSRVs following Chessa et al. (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Across Lebanese flocks the R2 retrotype defined by the presence of enJSRV-18 only and considered a marker of \\u0026ldquo;modern\\u0026rdquo; breeds, was the most frequent, occurring in 68.1% of Lebanese animals (Fig.\\u0026nbsp;5A).\\u003c/p\\u003e \\u003cp\\u003eThe R4 retrotype (enJSRV-18\\u0026thinsp;+\\u0026thinsp;enJSRV-7), commonly associated with Mediterranean populations, was the second most common retrotype in Lebanon (overall 14.6%). However, R4 showed marked spatial heterogeneity, varying from 0% to ~\\u0026thinsp;50% among herds (Fig.\\u0026nbsp;5B). R4 was significantly less frequent in north-eastern Lebanon (Baalbek, Hermel, Beqaa) and significantly more frequent and more evenly distributed among coastal and some south-eastern localities (including Rachaiya) on the Mediterranean side (Kruskal\\u0026ndash;Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.005377, \\u0026lt; 0.01). The two sampled Syrian herds exhibited a substantially higher R4 prevalence (\\u0026gt;\\u0026thinsp;50%), a difference that is highly significant when compared with Lebanese flocks (Kruskal\\u0026ndash;Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.000164).\\u003c/p\\u003e \\u003cp\\u003eBeyond R2 and R4, several other retrotypes were detected at lower frequencies. The RB retrotype (enJSRV-18\\u0026thinsp;+\\u0026thinsp;enJSRV-16) was the most frequent after R2/R4, with an overall prevalence of 10.6% in Lebanese Awassi. RB was not detected in Baalbek but reached 13.3% in Saida, 8.3% in Hermel, and 7.0% in the Beqaa; the highest RB frequency was observed in the Syrian Al-Qusayr herd (66.7%). The RA retrotype (enJSRV-18\\u0026thinsp;+\\u0026thinsp;enJSRV-15) occurred in 6.3% of Lebanese animals (Supplementary Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe R6 retrotype (enJSRV-18\\u0026thinsp;+\\u0026thinsp;enJSRV-22), which is more typical of northern European and some Caucasus populations, was comparatively rare in Lebanon (5.5%; 14 samples), and these R6 carriers were scattered across 14 different herds (Supplementary Figure S2). Retrotypes defined by combinations of three or more insertions were uncommon: for example, RF (18\\u0026thinsp;+\\u0026thinsp;7+16) was observed in ~\\u0026thinsp;1.6% of animals, while RD (18\\u0026thinsp;+\\u0026thinsp;15+16) was detected in ~\\u0026thinsp;0.4%. The four-locus combination RG (18\\u0026thinsp;+\\u0026thinsp;7+16\\u0026thinsp;+\\u0026thinsp;22) and RH (18\\u0026thinsp;+\\u0026thinsp;16+22) were each found in a single individual.\\u003c/p\\u003e \\u003cp\\u003eAt the herd level, retrotype richness was highest in the Kobayat commercial flock (northern Lebanon) and in the two Syrian flocks, each displaying at least five distinct retrotypes. This pattern is consistent with greater animal movement and commercial exchange in these herds and highlights how management practices influence local retrotype diversity. Overall, the retrotype distribution in Lebanese Awassi reflects a predominance of modern-type ancestry (R2) combined with measurable Mediterranean and regionally specific signatures (R4, RB, RA), shaped by both historical dispersal routes and contemporary flock management.\\u003c/p\\u003e\\n\\u003ch3\\u003eenJSRV-6 solo-LTR in Lebanese Awassi sheep\\u003c/h3\\u003e\\n\\u003cp\\u003eenJSRV-6, a deeply conserved endogenous retrovirus previously reported to be fixed across Ovis genomes (Arnaud et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e), was initially included here as an internal control for enJSRV genotyping. PCR assays targeting the 5\\u0026prime; and 3\\u0026prime; proviral ends, together with the empty-site PCR, produced an unexpected pattern in a subset of samples. Fifteen of the 277 tested animals yielded clear 5\\u0026prime; and 3\\u0026prime; LTR amplicons but lacked the corresponding 5\\u0026prime;/3\\u0026prime; provirus amplicons (Fig.\\u0026nbsp;6). In several additional samples the empty-site assay amplified a fragment of \\u0026asymp;\\u0026thinsp;750 bp rather than the expected\\u0026thinsp;\\u0026asymp;\\u0026thinsp;302 bp; Sanger sequencing of representative products confirmed that these anomalous bands correspond to solo long terminal repeat (solo-LTR) alleles derived from recombination between proviral LTRs (Mager \\u0026amp; Stoye, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Perucatti et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Verneret et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTaken together, the PCR and sequencing results indicate three genotypic classes at the enJSRV-6 locus: (i) homozygous for the full-length provirus (hereafter \\u0026ldquo;Homo/Prov\\u0026rdquo;); (ii) heterozygous, carrying one provirus allele and one solo-LTR allele (\\u0026ldquo;Hetero/Prov-LTR\\u0026rdquo;); and (iii) homozygous for the solo-LTR (\\u0026ldquo;Homo/LTR\\u0026rdquo;) (Fig.\\u0026nbsp;7). Density maps constructed from the 254 Lebanese samples (Syrian samples treated separately) show that the three genotypes are widespread: 47.64% Homo/Prov, 47.64% Hetero/Prov-LTR and 4.72% Homo/LTR (Fig.\\u0026nbsp;7; Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eTable summarizing the PCR results of enJSRV-6 in terms of zygosity for all the samples tested\\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=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eenJSRV-6\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eHomo/Pro\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eHetero/Pro-LTR\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eHomo/LTR\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eProvirus\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eNegative\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eSolo LTR\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eEmpty Locus\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNegative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003ePositive\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eNumber of samples\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e126\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e136\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e15\\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\\u003eThe geographic distribution of genotypes is non-random. The Hetero/Prov-LTR genotype is significantly enriched in southwestern Lebanon (Saida, Jezzine, Nabatieh) and is broadly distributed among flocks in that region (Kruskal\\u0026ndash;Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.000578; Fig.\\u0026nbsp;8A). Correspondingly, the combined frequency of animals carrying at least one solo-LTR allele (Hetero/Prov-LTR\\u0026thinsp;+\\u0026thinsp;Homo/LTR) is higher in these southern localities than elsewhere (Kruskal\\u0026ndash;Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.042320). Two herds in Nabatieh (total n\\u0026thinsp;=\\u0026thinsp;10) are extreme examples: none of the sampled individuals in these herds were Homo/Prov; all were either Hetero/Prov-LTR or Homo/LTR (Fig.\\u0026nbsp;8B).\\u003c/p\\u003e \\u003cp\\u003eThese findings indicate that (i) enJSRV-6 is present in all sampled genomes, consistent with an ancient integration event, and (ii) homologous recombination between LTRs has produced a frequent solo-LTR allele that has achieved substantial prevalence in several Lebanese flocks. The high frequency and partial homozygosity of the solo-LTR allele suggests that the recombination event is not recent and has been propagated through multiple generations. Geographic clustering of the solo-LTR allele may reflect historical founder effects, restricted gene flow, or local breeding practices; elucidating the relative contribution of these processes will require broader regional sampling and complementary autosomal data.\\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eMitochondrial DNA analysis provides a maternal perspective on the evolutionary history and demographic composition of livestock populations. In the Lebanese Awassi, Cyt-b sequence data revealed a high level of maternal diversity and the co-occurrence of four established sheep haplogroups: A (≈ 36%), B (≈ 41.5%), C (≈ 18%) and the rare E (≈ 4.5%). Haplogroups A, B and C are widely distributed and collectively represent the major maternal lineages in domestic sheep (Bruford et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Meadows et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). The near-codominance of A and B in Lebanon contrasts with some “improved” Awassi populations, which show a stronger bias toward haplogroup B (Meadows et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e) and indicates that the Lebanese Awassi herd has retained substantial ancestral maternal variation. The presence of haplogroup C at appreciable frequency, and the detection of the relatively uncommon haplogroup E (often associated with fat-tailed Middle Eastern breeds), further point to multiple maternal inputs into the Lebanese population. Collectively, these results are consistent with Lebanon’s historical role as a crossroads of animal movement and trade during and after the Neolithic, and with a history of repeated admixture rather than a single, strongly bottlenecked maternal origin.\\u003c/p\\u003e \\u003cp\\u003eThe enJSRV insertional polymorphisms provide a complementary, largely independent signal of sheep demographic history. In our sample enJSRV-18 was ubiquitous, and the R2 retrotype (enJSRV-18 alone) predominated (≈ 68%), consistent with a substantial contribution from lineages associated with the second wave of sheep dispersal that produced many modern breeds (Chessa et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). The Mediterranean R4 retrotype (enJSRV-18 + enJSRV-7) was also present (≈ 14.6%) and exhibited a clear geographic bias: it was more frequent in coastal and western localities (including historically important Phoenician ports) and comparatively rare in eastern highland regions (e.g. Baalbek, Hermel, Beqaa). This spatial pattern suggests that maritime and coastal trade routes have left a detectable genetic imprint on local flocks, whereas Lebanon’s central mountain ranges have acted as partial barriers to gene flow, producing contrasting retrotype compositions between coastal and inland flocks.\\u003c/p\\u003e \\u003cp\\u003eThe low but notable frequency of R6 (≈ 5.5%), a type more typical of northern Europe and some Caucasus populations (Chessa et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Schroeder et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e), indicates limited introgression from more distant sources, plausibly driven by commercial exchanges rather than historical dispersals. The Kobayat herd, a commercial flock with frequent livestock exchanges, displayed the highest retrotype diversity, which underlines the sensitivity of enJSRV profiles to recent management practices and animal movement. Overall, the combined mtDNA and enJSRV data portray a population shaped by both ancient demographic processes and ongoing anthropogenic-mediated gene flow.\\u003c/p\\u003e \\u003cp\\u003eA particularly noteworthy observation is the high prevalence of a solo-LTR allele at the enJSRV-6 locus: more than half of the tested animals carried a solo-LTR in at least one chromosomal copy (54.5% of samples), and a minority were homozygous for the solo-LTR. Solo-LTRs arise by homologous recombination between 5′ and 3′ LTRs of a provirus and accumulate over long evolutionary timescales (Mager \\u0026amp; Stoye \\u003cspan class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e, Perucatti et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Verneret et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The absence of empty pre-integration alleles for enJSRV-6 and the presence of solo-LTRs are consistent with an ancient insertion that predates the Capra–Ovis split and has since undergone recombination in certain lineages (Arnaud et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). The substantial frequency and partial homozygosity observed here indicate that the recombination event is not recent and has been transmitted through multiple generations.\\u003c/p\\u003e \\u003cp\\u003eBecause enJSRV-6 has been thought to be fixed across sheep, the detection of a frequent solo-LTR allele in Lebanese Awassi (and reported previously in some Turkish local breeds) suggests regional or lineage-specific evolutionary dynamics (Chessa et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Ayanoğlu, \\u003cspan class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Demirci et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). The geographic heterogeneity in solo-LTR frequency (e.g. enrichment in southern localities) could reflect historical founder effects, drift in relatively closed breeding systems, or selection at linked loci. Regardless of mechanism, the enJSRV-6 solo-LTR appears to be a stable genomic marker that may be useful for reconstructing deep phylogeographic relationships among Middle Eastern sheep populations, provided its distribution is assessed in broader comparative samples.\\u003c/p\\u003e \\u003cp\\u003eThe joint evidence from mtDNA and enJSRV retrotypes supports a scenario in which Lebanese Awassi sheep are the product of layered demographic events: an ancient substrate of regionally adapted stock, later admixture with populations carrying “modern” retrotypes, and ongoing local exchanges that introduce additional diversity. This mosaic is consistent with models of multiple dispersal waves during sheep domestication and subsequent historical translocations across the Mediterranean and Near East (Chessa et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Meadows et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFrom a conservation and management perspective, the high mitochondrial and retroviral diversity documented here is an asset. It argues for conservation strategies that preserve within-breed variability by avoiding indiscriminate crossbreeding and by maintaining representative herds from different geographic regions. Herds that retain uncommon mitolineages or rare retrotypes (and those harboring distinctive enJSRV-6 solo-LTR frequencies) merit targeted conservation attention and could serve as repositories for regional genetic variation.\\u003c/p\\u003e \\u003cp\\u003eSeveral limitations temper the inferences that can be drawn from the present study. Mitochondrial markers reflect only the maternal lineage and do not capture the autosomal or paternal genetic variation that shapes most phenotypic traits; likewise, enJSRV insertions provide a useful but partial record of historical events. To obtain a fuller picture of population structure and admixture, genome-wide autosomal SNP data and Y-chromosome markers should be incorporated in future work. Broader geographic sampling across neighboring countries (Syria, Jordan, Turkey) would contextualize the Lebanese patterns and test hypotheses about routes of gene flow. Finally, formal demographic modelling (e.g. using coalescent or approximate Bayesian computation approaches) and temporal sampling -where feasible- would permit more precise reconstruction of the timing and magnitude of admixture and expansion events.\\u003c/p\\u003e \\u003cp\\u003eThis study establishes a baseline genetic portrait of the Lebanese Awassi sheep, demonstrating considerable maternal and retroviral diversity and identifying an intriguing regional signature in the enJSRV-6 solo-LTR. These results refine our understanding of sheep population history in the Levant and provide concrete targets for conservation and future genomic investigation.\\u003c/p\\u003e\"},{\"header\":\"METHODS\",\"content\":\"\\u003ch2\\u003eSampling and DNA extraction\\u003c/h2\\u003e\\u003cp\\u003eBlood samples were collected from 254 presumably unrelated Awassi sheep in Lebanon (47 herds across 12 administrative departments) and from 23 sheep in two Syrian herds (Fig.\\u0026nbsp;1). Sampling was performed by licensed veterinarians from the Lebanese Agricultural Research Institute (LARI), with informed consent obtained from animal owners. All procedures were conducted in strict accordance with internationally accepted animal welfare standards, following the principles of the ARRIVE guidelines and relevant national regulations for the care and use of animals in research, ensuring minimal stress and discomfort to the animals. Genomic DNA was extracted from EDTA-anticoagulated whole blood using the GenElute™ Blood Genomic DNA kit (Sigma-Aldrich), following the manufacturer’s instructions. DNA concentration and purity were assessed by spectrophotometry. The sampling protocol and experimental procedures were reviewed and approved by the Research Ethics Committee (REC) of the Higher Center for Research (HCR) at the Holy Spirit University of Kaslik (USEK).\\u003c/p\\u003e\\n\\u003ch3\\u003eMitochondrial CytB DNA PCR and sequencing\\u003c/h3\\u003e\\n\\u003cp\\u003eA 1272-bp fragment of mitochondrial CytB (positions 14,078\\u0026ndash;15,349 of \\u003cem\\u003eOvis aries\\u003c/em\\u003e reference genome AF010406) was amplified using primers cytbF and cytbR (Meadows et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). PCR reactions were 50 \\u0026micro;L volume, containing: 1\\u0026times; Taq buffer (with 1.5 mM MgCl₂), 200 \\u0026micro;M of each dNTP, 0.5 \\u0026micro;M of each primer, and 1 U Taq DNA Polymerase (Qiagen Taq PCR Core Kit). Cycling conditions were: initial denaturation at 95\\u0026deg;C for 5 min; 40 cycles of 95\\u0026deg;C for 30 s, 55\\u0026deg;C for 30 s, 72\\u0026deg;C for 1 min; and a final extension at 72\\u0026deg;C for 10 min. PCR products were verified by agarose gel electrophoresis (1% agarose, ethidium bromide) and purified with the GenElute\\u0026trade; PCR Clean-up Kit (Sigma-Aldrich).\\u003c/p\\u003e \\u003cp\\u003eCycle sequencing of both strands was performed using the BigDye\\u0026reg; Terminator v1.1 kit (Applied Biosystems) under standard conditions. Labeled products were run on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequences were assembled and edited in ChromasPro v.1.7.5.4 (Technelysium). Ambiguous bases at read ends were trimmed, yielding a final alignment of 1034 bp per sample.\\u003c/p\\u003e \\u003cp\\u003eFor the data analysis, indices of sequence variation, including the total number of polymorphic sites, nucleotide diversity (π), mean number of nucleotide differences (D), and average number of nucleotide substitutions per site (K), were calculated using DnaSP v6.0.73 (Rozas et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Haplotype structure and diversity parameters were also estimated using the same software.\\u003c/p\\u003e \\u003cp\\u003eA median-joining (MJ) network of the mitochondrial \\u003cem\\u003ecytochrome b\\u003c/em\\u003e (Cyt-b) region was constructed with NETWORK v4 (Bandelt et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e) to infer relationships among Awassi haplotypes. The network was built from the Cyt-b sequences generated in this study together with ten domestic reference sequences representing the five major mitochondrial haplogroups previously described in sheep (Meadows et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e): Haplogroup A (HM236174, HM236175), Haplogroup B (HM236176, HM236177), Haplogroup C (HM236178, HM236179), Haplogroup D (HM236180, HM236181), and Haplogroup E (HM236182, HM236183).\\u003c/p\\u003e \\u003cp\\u003eThe resulting network allowed visualization of mutational steps and genealogical relationships among Lebanese Awassi haplotypes and their connection to global \\u003cem\\u003eOvis aries\\u003c/em\\u003e mitochondrial lineages.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDNA amplification of enJSRV\\u003c/h2\\u003e \\u003cp\\u003eThe extracted genomic DNA was also used as a template for the detection of endogenous retroviruses. The presence of seven enJSRV-6, -7, -8, -15, -16 and \\u0026minus;\\u0026thinsp;18 and \\u0026minus;\\u0026thinsp;22 was evaluated by PCR as already described by (Arnaud et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). The amplification of each enJSRV provirus was obtained by two sets of PCR reactions (5\\u0026rsquo;- and 3\\u0026rsquo;-PCR). The presence of solo LTRs was also assessed, for each provirus, using three distinct PCR assays specifically designed to amplify solo LTRs and the empty genomic DNA around the proviral integration site. All the primers used in this study are presented in supplementary fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. Among the seven enJSRVs, enJRSV-6 is the oldest and used as a positive control of enJSRV amplification while the six other enJSRVs are insertionally polymorphic within the \\u003cem\\u003eOvis aries\\u003c/em\\u003e genomes (Arnaud et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe reactions were carried out in a final volume of 50\\u0026micro;l, containing 5\\u0026micro;l of DNA template, 1x Taq Buffer (Tris-Cl, KCl, (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, 1.5 mM MgCl\\u003csub\\u003e2\\u003c/sub\\u003e; pH 8.7), 1x Q solution, 200\\u0026micro;M of each dNTP, 2.5U of HotStar Taq ADN Polymerase (\\u0026laquo; HotStar Taq\\u0026reg; DNA Polymerase \\u0026raquo;, Qiagen, Germany) et 0.2\\u0026micro;M of each primer.\\u003c/p\\u003e \\u003cp\\u003eRetrotypes for each animal were assigned based on the presence/absence profile of enJSRV insertions following Chessa et al. (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e): R2 (only enJSRV-18 present), R4 (enJSRV-18 and \\u0026minus;\\u0026thinsp;7), R6 (enJSRV-18 and \\u0026minus;\\u0026thinsp;22), R11 (enJSRV-18 and \\u0026minus;\\u0026thinsp;8), as well as two novel categories defined here: RA (enJSRV-18 + -15) and RB (enJSRV-18 + -16). Other combinations involving three or more proviruses were noted but were rare.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study involved non-experimental field sampling of client-owned animals. Blood collection was performed by licensed veterinarians from the Lebanese Agricultural Research Institute (LARI) in compliance with international guidelines for the ethical treatment of animals, including the International Council for Laboratory Animal Science (ICLAS) ethical principles for animal research. Sampling procedures did not involve anesthesia, euthanasia, or experimental interventions. In accordance with GSE policies for studies involving client-owned animals, informed consent was obtained from all animal owners prior to sampling, and all veterinary procedures adhered to recognized best practices. Under Lebanese regulations (non-experimental veterinary sampling of domestic livestock), this work did not require formal approval from an animal ethics committee or institutional review board; accordingly, no approval number is issued.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Not applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe mitochondrial cytochrome \\u003cem\\u003eb\\u003c/em\\u003e sequences generated in this study have been deposited in the GenBank nucleotide database (INSDC) under accession numbers \\u003cstrong\\u003ePX994374\\u0026ndash;PX994861\\u003c/strong\\u003e. Public release of the records will be on 18 Feb, 2026.\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional raw data supporting the findings of this study, including detailed genotyping matrices and supplementary metadata, are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was cofounded by the Lebanese Agricultural Research Institute (LARI) and Ecole Pratique des Hautes Etudes (EPHE)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026rsquo; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: Christophe Terzian, Alain Abi-Rizk\\u003cbr\\u003e\\u0026nbsp;Project administration: Frederic Arnaud\\u003cbr\\u003e\\u0026nbsp;Investigation: Jeanne El Hage\\u003cbr\\u003e\\u0026nbsp;Formal analysis \\u0026amp; methodology: Jeanne El Hage, Fr\\u0026eacute;d\\u0026eacute;rick Arnaud, Fr\\u0026eacute;d\\u0026eacute;ric Boyer, Barbara Viginier, Fran\\u0026ccedil;ois Pompanon,\\u0026nbsp;\\u003cbr\\u003e\\u0026nbsp;Data interpretation: Jeanne El Hage, Fr\\u0026eacute;d\\u0026eacute;rick Arnaud, Fr\\u0026eacute;d\\u0026eacute;ric Boyer\\u003cbr\\u003e\\u0026nbsp;Supervision: Fr\\u0026eacute;d\\u0026eacute;rick Arnaud, Alain Abi-Rizk\\u003cbr\\u003e\\u0026nbsp;Writing \\u0026ndash; review \\u0026amp; editing: All authors\\u003cbr\\u003e\\u0026nbsp;Final approval of manuscript: All authors\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlberto, F. 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Selection Evol.\\u003c/em\\u003e \\u003cb\\u003e38\\u003c/b\\u003e (5), 513\\u0026ndash;524. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1186/1297-9686-38-5-513\\u003c/span\\u003e\\u003cspan address=\\\"10.1186/1297-9686-38-5-513\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e (2006).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVerneret, M., Lannes, B., Arnaud, F. \\u0026amp; Palmarini, M. A genome-wide study of ruminants uncovers two endogenous retrovirus families with recent activity. \\u003cem\\u003eMobile DNA\\u003c/em\\u003e (2024). (Advance online publication).\\u003c/span\\u003e\\u003c/li\\u003e \\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Awassi sheep, genetic diversity, mitochondrial DNA, endogenous Jaagsiekte sheep retrovirus, population structure, Middle East domestication\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8807670/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8807670/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe Awassi is a native sheep breed of Lebanon with high historical and economic value. Despite its importance, its genetic background has not been characterized, leaving limited understanding of its origins, diversity, and regional connections.\\u003c/p\\u003e \\u003cp\\u003eThis study aimed to characterize the genetic diversity and population structure of Awassi sheep from Lebanon and neighboring Syria using two complementary genetic systems that trace distinct evolutionary histories: maternal mitochondrial DNA and insertional polymorphisms of endogenous Jaagsiekte sheep retroviruses.\\u003c/p\\u003e \\u003cp\\u003eGenetic analyses were conducted on two hundred seventy-seven animals. Sequencing of a one thousand thirty-four base pair fragment of the mitochondrial cytochrome b gene identified eighty-six maternal haplotypes that clustered into four haplogroups (A, B, C, and E). Maternal haplotype diversity was high, and most individuals belonged to haplogroups A and B, indicating contributions from both western and eastern maternal lineages.\\u003c/p\\u003e \\u003cp\\u003eRetroviral insertion profiling showed that the retroviral type carrying only endogenous Jaagsiekte sheep retrovirus element eighteen was predominant. A Mediterranean retroviral type carrying both endogenous Jaagsiekte sheep retrovirus eighteen and seven was also present, especially in coastal flocks, suggesting ancient gene flow through maritime trade. Other retroviral combinations were rare.\\u003c/p\\u003e \\u003cp\\u003eAnalysis of endogenous Jaagsiekte sheep retrovirus six revealed a high frequency of solo long terminal repeat alleles, indicating an ancient recombination event. Approximately half of the animals were heterozygous for this allele and some homozygous. No pre-integration alleles were detected, confirming fixation of endogenous Jaagsiekte sheep retrovirus six in domestic sheep.\\u003c/p\\u003e \\u003cp\\u003eLebanese Awassi sheep exhibit substantial genetic diversity across both maternal and retroviral systems, reflecting a complex population history shaped by multiple lineages and regional exchanges. This study provides the first comprehensive genetic characterization of this native breed and contributes to broader understanding of sheep domestication in the Middle East, while supporting evidence-based conservation of this valuable genetic resource.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Mitochondrial and Retroviral Markers Reveal High Genetic Diversity and Regional Structure in the Lebanese Awassi Sheep\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-04 15:34:35\",\"doi\":\"10.21203/rs.3.rs-8807670/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-18T20:53:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"56359097188816443295114088943584093700\",\"date\":\"2026-05-15T15:25:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"217754957252306613485245657373667462311\",\"date\":\"2026-05-15T10:52:13+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-04-21T03:27:21+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"47547578526122460566844507852533243200\",\"date\":\"2026-04-03T01:12:21+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"233761564089744947276427164653576375560\",\"date\":\"2026-04-01T14:49:38+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-03-02T07:27:39+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-03-02T07:17:50+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-02-24T14:45:54+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-02-18T09:35:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2026-02-18T09:31:11+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"9e0e79c1-4a93-4910-8171-b412502e01f1\",\"owner\":[],\"postedDate\":\"March 4th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-18T20:53:41+00:00\",\"index\":53,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"56359097188816443295114088943584093700\",\"date\":\"2026-05-15T15:25:11+00:00\",\"index\":52,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"217754957252306613485245657373667462311\",\"date\":\"2026-05-15T10:52:13+00:00\",\"index\":51,\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":63765096,\"name\":\"Biological sciences/Evolution\"},{\"id\":63765097,\"name\":\"Biological sciences/Genetics\"}],\"tags\":[],\"updatedAt\":\"2026-03-04T15:34:35+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-04 15:34:35\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8807670\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8807670\",\"identity\":\"rs-8807670\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}