Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep

Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed 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 Research Article Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep Lei Gao, Yiyuan Zhang, Jingquan Yang, Zhenliang Zhang, Yucheng Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6146587/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tail fat in sheep ( Ovis aries ), has evolved mainly in response to cold weather for better energy storage. As things stand, too much tail fat in sheep can lead to a reduction in feed utilisation and is also unpopular with consumers due to the excessive fat content in the tail of sheep. Therefore, the need to find the mechanism of tail fat formation is obvious. In this study, we elected to utilise Kazakh sheep, prolific Suffolk sheep, and their hybrid F2 generation as research objects. Sheep transcriptome sequencing technology was employed to screen and explore target candidate genes related to sheep tail fat deposition. Comparison with RNA-seq data from fat-tailed and thin-tailed tissue, the LncRNA-mRNA-miRNA axis was identified as main functional pathway in the formation of fat in tail. Our results offer valuable insights into the fat deposition of sheep and provide a significant genomic resource for future genetic studies and the enhancement of genome-assisted breeding in sheep and other domestic animals. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Fats are mainly composed of triacylglycerols (TAGs), which function as the primary storage medium for energy in animals [ 1 – 4 ]. In circumstances where nutrients are plentiful, excess nutrients are transferred to fat reserves, which are then stored in the liquid phase of fat [ 5 – 9 ]. These fat reserves provide essential sustenance during periods of food scarcity [ 10 , 11 ]. One of the most effective strategies for reducing energy expenditure is the reduction of fat, a process which has a substantial impact on the quality of meat produced by livestock [ 1 , 12 ]. A significant disparity exists in the capacity of various sheep breeds to deposit fat in the tail [ 13 ]. In light of these findings, sheep can be categorised into two distinct groups: fat-tailed sheep, which include breeds such as the Kazakh and Altay sheep, and thin-tailed sheep, which include the Chinese Merino and Suffolk sheep, among others [ 14 ]. The fat-tailed trait in sheep, specifically the rump, is a biological trait that is essential for survival in challenging environments and has played a pivotal role in specific historical periods [ 15 – 17 ]. The fat-tailed trait of northern Chinese sheep is the result of long-term evolution and selection under complex and harsh natural environmental conditions [ 18 , 19 ]. These conditions have endowed the breeds with strong adaptability to the local severe natural ecological environment, including excellent characteristics such as tolerance to coarse feed, strong stress resistance, rapid growth and development, and high meat and fat production performance [ 19 ]. Some of them evolve a strategy in sheep, which is deposit the fat into their tails with unknown reasons [ 20 ]. The Kazakh sheep (HUS) is named after its round and fat tail, which is full of fat, and its main characteristics are coarse wool, big tail, and both meat and fat [ 21 , 22 ]. On the contrary, tail fat deposition capacity of prolific Suffolk sheep (SFK) differs greatly between fat-tailed and lean-tailed sheep, but the genetic characteristics and molecular mechanisms of fat deposition are still unclear. Previous studies had explored the genetic mechanism of the fat tail formation by whole-genome sequencing [ 23 ]. Molecular markers such as SNP and CNV, are used to define some candidate genes which related to the phenotypes [ 24 ]. Meanwhile, a set of gene locus are detected by the obvious discrepancy of allele frequency. It is reported that a set of RNAs, such as miRNAs and lncRNAs, play an important role in regulating the fat deposition [ 25 ]. Wang, et al . found the fat deposition related gene PDGFD has alternative splicing function, led to relevant with tail fat deposit [ 15 , 26 ]. Transcription regulator IRF2BP2 also represented allele differential expression in muscle tissue, resulting a vital role in muscle growth process [ 26 , 27 ]. In this study, we are trying to explore the mechanism of fat deposit with transcriptome sequencing. We bred the F2 generation of prolific Suffolk and Kazakh sheep, and extract the caudal fat tissue for sequencing samples. A cluster of RNAs have been detected that participate in the metabolic pathways, which impact the adipogenesis and tail lipid formation. We also defined a group of miRNAs and lncRNAs which are differential expressed in fat tissue. Those findings provide an opportunity for us to further look inside in the caudal fat formation. MATERIALS AND METHODS Sample preparation Three healthy adult Kazakh sheep (HUS) which is fat-tailed, F2 generation (F2) of crossbreeding between Kazakh (HUS) and Suffolk sheep (SFK) and adult Suffolk sheep, lean-tailed, of nearly the same age were selected and uniformly reared and managed. Tail tissue samples are collected in December when tail fat deposition is at its highest. Tail fat samples were collected in the shortest possible time using specially treated surgical instruments for RNA experiments (all from the same side), frozen in liquid nitrogen and stored in a refrigerator at -80℃ for back-up. RNA isolation RNA was extracted from tail tissues, and the samples underwent stringent quality control based on the following criteria. First, agarose gel electrophoresis was used to assess RNA integrity and detect potential DNA contamination. Second, RNA concentration and purity were preliminarily quantified. Finally, the integrity of RNA was accurately measured. Library preparation For library construction, strand-specific libraries were prepared using a ribosomal RNA (rRNA) depletion method. Total RNA was first treated to remove rRNA, and the remaining RNA was fragmented into short segments of 250–300 bp. These fragments served as templates for first-strand cDNA synthesis using random oligonucleotide primers. The second-strand cDNA was then synthesized using dNTPs as substrates. The double-stranded cDNA was purified, end-repaired, A-tailed, and ligated with sequencing adapters. The second strand of cDNA was degraded using the USER enzyme, and the remaining cDNA was amplified by PCR to obtain the final strand-specific library. To ensure library quality, a Qubit fluorometer was used for preliminary quantification, diluting the library to 1 ng/µL. The Agilent 2100 Bioanalyzer was then employed to assess the insert fragment size, which was expected to range from 250 to 300 bp. After confirming the expected insert size, qPCR was conducted for precise quantification of the library's effective concentration, which was required to exceed 2 nM to ensure high-quality samples. Finally, libraries that passed quality control were pooled based on their effective concentration and sequencing data requirements for Illumina PE150 sequencing. In paired-end sequencing (PE150), each end of the cDNA insert is sequenced to a length of 150 bp, with the insert fragment serving as the sequencing unit. Paired-end sequencing not only provides sequence information from both ends of the insert fragment but also offers insights into the length between the two ends, facilitating downstream assembly and alignment. RNA sequencing The sequencing was performed using the Sequencing by Synthesis (SBS) method. In this approach, the flow cell was loaded with four fluorescently labeled dNTPs, DNA polymerase, and adapter primers. During each cycle of extension, a fluorescently labeled dNTP was incorporated into the complementary DNA strand, releasing a fluorescence signal specific to the incorporated nucleotide. These signals were captured by the sequencer, and computational software converted the fluorescence data into sequencing peaks, providing the nucleotide sequence of the target fragments. Data analysis RNA-seq data is applied quality control and adapter trimming by Trim Galore v0.6.4_dev and mapped to the reference genome using STAR v2.7.2d. And we performed reads summarization for genomic features via featureCounts v2.0.0. The raw read counts normalization and differential expression were determined by DESeq2 v1.34.0. The number of reads matched to an expressed gene was standardized as fragments per kilobase of exon per million mapped fragments (FPKM) values. The genes that exhibited |log2(fold change)| ≥ 2 and adjusted P ≤ 0.05 in the comparisons between fat-tailed/fat-rumped and thin-tailed individuals were considered as differentially expressed genes. GO enrichment analysis used the enrichGO function provided within the R package clusterProfiler v4.2.2. RESULTS F2 generation of the thin- and fat-tailed sheep Sheep tail fat deposition are economically important traits and have been hot research topics in recent years [17]. Tail fat can satisfy the early human demand for lipids, and with the improvement of living standards, the demand for high-fat meat decreases [15]. Therefore, it is of great significance to explore the important genes regulating tail fat deposition and muscle growth rate in sheep to breed low-fat and high-yield meat sheep. In order to identify the key regulator of fat deposition, a breeding program was initiated. This involved the generation of F2 offspring from thin-tailed Suffolk sheep and fat-tailed Kazakh sheep (Fig. 1D). The Kazakh sheep is known as fat-tailed (Fig. 1A-B), whereas the Suffolk sheep have thin-tailed phenotype (Fig. 1C). The caudal fat tissue of the Kazakh and F2 was extracted, and it was found that the Kazakh tissue was larger than that of the F2 tail (Fig. 1E). However, the regulatory mechanism is still unclear. Transcriptomic analysis reveals potential regulatory modules To define the reconcile capability, we utilized the RNA sequencing to monitor gene expression dynamics using the Kazakh, Suffolk and F2 tail fat tissue. The FPKM distribution were calculated and showed an average level (Fig. 2A, Supplementary Table 1). Then, we compared the gene expression values and detected the significant up-regulated and down-regulated genes (Fig. 2B, Supplementary Table 2), such as PDGFD and IRF2BP2 which are consistent of previous studies. There are 1,049 up-regulated genes and 451 down-regulated genes in Kazakh sheep (HUS) than the F2 generation (CSH). A comparison was made between HUS and SFK with 754 genes that are expressed at high levels and 711 that are expressed at low levels. SFK has 830 up-regulated expressed genes compared to CSH, and 602 down-regulated genes. As previously mentioned, the identification of potential regulatory genes can be achieved through the analysis of the set of genes exhibiting differential expression levels in fat tails in comparison to thin tails (Fig. 2C). To further identify regulatory genes, we overlapped the differential expression genes. We compared each of the three groups, HUS, SFK and CSH, and further narrowed it down based on the overlap of differentially expressed genes (Fig. 3A-D), 93 genes are defined as potential key effectors in lipid metabolism. The Pearson correlation reflects the linear relationship among those three groups, which prove the robust (Fig. 3E). To specify the fat-tail regulated genes, we overlapped the high expressed genes in HUS and SFK, which compared with CSH (Fig. 3F). There are 36 genes up-regulated in HUS and SFK, and GO enrichment analysis results showed 5 core genes ( BDH1, EPHX1, BCAT2, FASN, ACACA ) participate in lipid metabolic process and fatty acid biosynthetic process (Fig. 3H-G). Based on previous analysis, we could conclude that the high-throughput sequencing data making it suitable for analyzing the regulatory modules with biological differences across groups and we defined 5 candidate genes could play an important role in mediating the formation of fat tail. Functional signature analysis of coding and non-coding RNAs Recognizing that function of non-coding RNAs is common challenge in RNA-seq data. For the past few decades, lncRNA have been regarded as transcripts of minimal biological significance [2, 16]. Nevertheless, there is mounting evidence to suggest that lncRNA is widespread in eukaryotes and plays a crucial role that regulated gene expression, particularly in mammals [11, 28]. Here, we attempt to explore the mechanism of fat tail formation by Gene Ontology (GO) analysis of the differential expressed lncRNA and around mRNA which are co-located in genome (Fig. 4A-C, Supplementary Table 3). First, we analyzed the mRNAs which may regulated by highly-expressed lncRNA in HUS than SFK mainly enriched in response to stimulus or stress, immune system process and cell-cell adhesion including protein binding function (Fig. 4A). By comparing the HUS with CSH, those genes participated in response to stress, extracellular region and receptor binding (Fig. 4B). Through the result of SFK and CSH, response to stimulus or stress and cytokine receptor binding represent the signal transduction pathways could be the potential executive processes (Fig. 4C). To further identify the functional elements involved in tail fat deposition, differential expressed genes (DEGs) were annotated to multiple pathways, which help us to find the unique program modulating the fat deposition in sheep tail. KEGG results shows that DEGs are enriched in Metabolic pathways, TCA cycle, Fatty acid metabolism, Fat digestion and absorption, Fatty acid biosynthesis (Fig. 5, Supplementary Table 4). These results revealed the significant genes controls the fat deposit in tail by participating fat-related pathways, and offered us an opportunity to declare the principle of fat deposition. Novel miRNA identification Small RNA (sRNA) idiosyncratically recognized and bound the RNA-induced silencing complex (RISC) and inhibited gene expression [29, 30]. sRNAs play an important role in regulating almost all events at the cellular level, such as individual development, cell proliferation and differentiation, tumor development and antiviral resistance, and are important members of the highly complex RNA regulatory network in cells, especially miRNA [22, 31]. To gain further insight into whether miRNA have any functional role in fat deposition, we evaluated a set of novel miRNAs and divide them into four subclusters according to the its relative expression levels (Fig. 6A-B, Supplementary Table 5). Finally, we examined the total differential expressed miRNAs and defined up-regulated miRNAs ( novel_401, novel_72, novel_120, novel_440, novel_85, novel_171, oar-miR-218a, novel_74, novel_144, oar-miR3958-3p, novel_107, novel_51, novel_46, oar-miR-543-3p ) in fat-tailed samples (Fig. 6C). These findings underline the miRNAs perhaps assume a vital role in fat tail deposition as epigenomic regulators. Furthermore, our RNA-seq dataset provides a unique platform to investigate sheep specific genes in fat deposition of tail, identifying regulatory modules, such as protein-coding genes, lncRNAs and miRNAs. Although most of these miRNAs are novel detected and those function remains to be further investigated. In summary, our study shows that mRNA could be regulated by lncRNAs in cold environment which is response to stress and be part of lipid metabolism pathways leading fat tail. And miRNAs as epigenomic regulator controls the fat-deposit-regulated genes expression level in fat tissue, to which leads the deposition in tail. DISCUSSION This study aimed to explore the genes regulating tail fat deposition and muscle growth rate in sheep, with the ultimate goal of breeding low-fat and high-yield meat sheep. The findings from the F2 generation of thin-and fat-tailed sheep crosses, along with transcriptomic, functional signature, and miRNA analyses, have provided valuable insights into the complex molecular mechanisms underlying tail fat deposition. The transcriptomic analysis using RNA sequencing of tail fat tissues from Kazakh, Suffolk, and F2 sheep was a crucial step. The identification of differentially expressed genes (DEGs) between these groups has shed light on potential regulatory genes. The large number of up-regulated and down-regulated genes in Kazakh sheep compared to the F2 generation, as well as between other pairwise comparisons, indicates significant genetic differences contributing to tail fat deposition. The overlapping of DEGs across the three groups (HUS, SFK, and CSH) to define 93 potential key effectors in lipid metabolism is a significant finding. These genes are likely to be central to the regulatory network controlling fat deposition in the sheep tail. The Pearson correlation among the groups validates the reliability of the high-throughput sequencing data, ensuring that the identified regulatory modules are robust and biologically relevant. The genes such as PDGFD and IRF2BP2 , which were consistent with previous studies, further support the validity of our approach and the potential importance of these genes in fat deposition. The exploration of the functions of non-coding RNAs, especially lncRNAs, through GO analysis, has revealed new aspects of fat tail formation. The finding that mRNAs potentially regulated by highly-expressed lncRNAs in Kazakh sheep compared to Suffolk sheep are enriched in processes such as response to stimulus or stress, immune system processes, and cell-cell adhesion is intriguing. This suggests that these biological processes may be indirectly related to fat deposition, perhaps through their effects on the overall physiological state of the tissue. The GO analysis comparing different groups also showed that genes were involved in various processes related to stress response and receptor binding. These results imply that the regulation of fat deposition in the sheep tail is a complex process, with multiple biological pathways and molecular functions interacting. The enrichment of DEGs in metabolic pathways, TCA cycle, fatty acid metabolism, fat digestion and absorption, and fatty acid biosynthesis as revealed by KEGG analysis further emphasizes the role of these canonical metabolic pathways in tail fat deposition. Identifying these pathways provides a framework for understanding how genes control fat deposition in the tail and offers potential targets for genetic manipulation in breeding programs. The identification of novel miRNAs and their classification into sub-clusters based on relative expression levels is an important addition to our understanding of fat deposition regulation. The up-regulated miRNAs in fat-tailed samples, such as novel_401, novel_72, and oar-miR-218a , suggest that these miRNAs may act as epigenomic regulators in fat tail deposition. miRNAs are known to play crucial roles in post-transcriptional gene regulation, and their involvement in fat deposition indicates a complex regulatory network at the miRNA level. Although most of these miRNAs are newly detected and their functions remain to be fully investigated, our results provide a starting point for future research. Understanding how these miRNAs interact with other regulatory elements, such as lncRNAs and protein-coding genes, will be essential for a comprehensive understanding of the molecular mechanisms of fat deposition in sheep tails. Overall, our study has integrated multiple levels of molecular analysis to identify potential regulatory elements in sheep tail fat deposition. The identification of coding genes, lncRNAs, and miRNAs involved in this process provides a rich resource for further research. Future studies could focus on validating the functions of the identified genes and miRNAs through in vitro and in vivo . For example, gene knockout or overexpression studies in sheep or relevant cell models could be used to directly assess the impact of these genes on fat deposition. Furthermore, understanding the interactions between different regulatory elements, such as how lncRNAs regulate mRNAs and how miRNAs fine-tune the expression of fat-deposition-related genes, will be crucial [ 29 , 32 ]. This knowledge could be applied to develop new breeding strategies to manipulate tail fat deposition, aiming to produce sheep with lower fat content in the tail while maintaining high-quality meat production. In conclusion, our study has laid a solid foundation for future research in this area, with potential implications for the sheep breeding industry. Declarations Funding This work was supported by the following grants: The Science and Technology Innovation Talents Project of Corp (2023CB007-03), The National Nature Science Foundation of China (31660651), China Agriculture Research System (CARS-39-07), Young Science and Technology Top Talent Program of Tianshan Talent Training Program in Xinjiang Province (2022TSYCCX0124), Xinjiang Agriculture Research System (XJARS-09-26), Project of Corps Science and Technology in Key Areas (2024AB017). Author Contribution L.G. performed the majority of the experiments, collected and analyzed the data, and drafted the initial manuscript. Y.Z. and J.Y. assisted in experimental work, data analysis, and interpretation, and helped in drafting the manuscript. Z.Z. conducted statistical analyses and prepared data visualizations. Y.L. and J.W. contributed to sample collection and conducted laboratory experiments. L.W. and P.W. contributed to the study design, provided essential resources, and offered critical feedback during manuscript preparation. Z.Z. conceived and designed the study, supervised the overall project, and provided critical revisions to the manuscript. Data Availability RNA-seq data have been deposited in the GEO database (GEO: GSE290403) and are publicly available as of publication. This study does not report original algorithms. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Ethics Statement Ethics Committee Approval: This study was approved by the Ethics Committee (Approval Number: A2024-008). References Sahu, B., et al., Role of Distinct Fat Depots in Metabolic Regulation and Pathological Implications. Rev Physiol Biochem Pharmacol, 2023. 186 : p. 135-176. Bakhtiarizadeh, M.R. and S.A. Salami, Identification and Expression Analysis of Long Noncoding RNAs in Fat-Tail of Sheep Breeds. 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Han, J., et al., Quantitative proteomic analysis identified differentially expressed proteins with tail/rump fat deposition in Chinese thin- and fat-tailed lambs. PLoS One, 2021. 16 (2): p. e0246279. Additional Declarations No competing interests reported. Supplementary Files S1.mRNAgene.FPKM.xlsx S2.DEGs.xlsx S3.lncRNAmRNAcolocationGOresults.xlsx S4.DEGsKEGGresults.xlsx S5.miRNADEresults.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-6146587","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":424287824,"identity":"991e9616-6e78-492e-99ac-cbdd22155748","order_by":0,"name":"Lei Gao","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Gao","suffix":""},{"id":424287825,"identity":"4869164f-86e0-4423-bc17-b1e5a057d385","order_by":1,"name":"Yiyuan Zhang","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Yiyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":424287826,"identity":"f1b72f5a-bae7-4ed2-875c-6958f7760fbb","order_by":2,"name":"Jingquan Yang","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Jingquan","middleName":"","lastName":"Yang","suffix":""},{"id":424287827,"identity":"ab0f902a-310e-4781-94af-4961f7352379","order_by":3,"name":"Zhenliang Zhang","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Zhenliang","middleName":"","lastName":"Zhang","suffix":""},{"id":424287828,"identity":"2800d46e-2af3-4945-86e4-a771a1f185a3","order_by":4,"name":"Yucheng Liu","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Yucheng","middleName":"","lastName":"Liu","suffix":""},{"id":424287829,"identity":"5198f216-0997-40c4-95a4-ef34a0154fb9","order_by":5,"name":"Jingjing Wang","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Wang","suffix":""},{"id":424287830,"identity":"3e4e7b27-3fa8-47d7-8d93-4813c4affafe","order_by":6,"name":"Limin Wang","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Wang","suffix":""},{"id":424287831,"identity":"f330b24c-c568-4aed-84eb-4d5f38a91f61","order_by":7,"name":"Pengcheng Wan","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"prefix":"","firstName":"Pengcheng","middleName":"","lastName":"Wan","suffix":""},{"id":424287832,"identity":"8e519f60-7b0d-4128-b911-fe4d1be87b38","order_by":8,"name":"Zongsheng Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACCTiD+QAzhJVAtBa2BJK18BgQp0V+dvPDhz8q7tjNn93z+XPBn8MM/Ow5Bgw/d+DWwjjnmLGBxJlnyRvunN0mPYPnMINkzxsDxt4zuLUwSySYSRi2HU42kMjdxswjcZjB4EaOATNjG24tbBLp3yQSgVrkZ+Q8/sxjcJjBnpAWHokcM4mDbYftGG7kMEjzJABtkSCgRUIip9iw4czhBIMbaWbSPAfSeYAeKzjYi0eL/Iz0jcAQO2wvPyMZ6LA/1nL87ckbH/zEowUGEhtgLgURBwhrYGCwJ0bRKBgFo2AUjFAAAICDTshA1NwSAAAAAElFTkSuQmCC","orcid":"","institution":"Shihezi University","correspondingAuthor":true,"prefix":"","firstName":"Zongsheng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-03-03 13:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6146587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6146587/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77855869,"identity":"7c146d8c-a468-44ab-8cc8-d51bd3da5d04","added_by":"auto","created_at":"2025-03-06 07:42:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":716323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent phenotypes of the sheep (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOvis aries\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) and its fat deposition in tail\u003c/strong\u003e. \u003cstrong\u003eA-B\u003c/strong\u003eThe Kazakh sheep, fat-tailed sheep, is characterized by an increased propensity for fat deposition in the tails. \u003cstrong\u003eC\u003c/strong\u003e Suffolk sheep, thin-tailed sheep, is endowed with a diminutive tail size. \u003cstrong\u003eD\u003c/strong\u003e F2 generation of Kazakh and Suffolk sheep, exhibit a thin tail characteristic. However, they demonstrate a higher deposition rate in comparison to Suffolk sheep. \u003cstrong\u003eE\u003c/strong\u003e The adipose tissues of the F2 and Kazakh sheep breeds were analyzed. The left side corresponds to the F2, while the right side is representative of the Kazakh sheep.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/8069ff59450690d02c998cf6.png"},{"id":77855870,"identity":"e70a91e3-0553-422a-9964-ecc30fead35c","added_by":"auto","created_at":"2025-03-06 07:42:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":364803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome-wide analysis based on the differential fat deposition groups\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e FPKM distribution levels are indicative of RNA expression levels in the tails' tissues across different samples. \u003cstrong\u003eB\u003c/strong\u003e The volcano plot is employed to illustrate the gene expression between the parents and the F2 or within those patents. The red segments of the plot denote up-regulated genes, while the green segments represent down-regulated genes. The X-axis denotes the fold change, while the Y-axis indicates the p-value. \u003cstrong\u003eC\u003c/strong\u003e The heatmap illustrates the variance in RNA levels among the sheep. The red denotes elevated levels, whereas the blue indicates reduced levels.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/79b8680d6debb246df4a42d7.png"},{"id":77855871,"identity":"91b9bd7f-7d09-4fa9-aea1-8f575a6b10d5","added_by":"auto","created_at":"2025-03-06 07:42:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":504477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVenn diagrams summarize unique and common variants among groups.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e Two-circle Veen diagrams shows the shared and unique differential expression genes among the HUS, SFK and CSH. \u003cstrong\u003eD\u003c/strong\u003e The three-circle Veen diagram reveals a marked change in 93 genes among all samples. \u003cstrong\u003eE\u003c/strong\u003e Pearson correlation heatmap demonstrates that the variation between the replicate samples is minimal, which valid the duplicates. \u003cstrong\u003eF\u003c/strong\u003eThe Veen diagrams of up-regulated genes in HUS and SFK, 36 genes were detected via overlapping. \u003cstrong\u003eG\u003c/strong\u003e Heatmap of 36 up-regulated genes that co-located in both samples. \u003cstrong\u003eH\u003c/strong\u003e GO enrichment of the 36 up-regulated genes, which concludes biology process (BP), cellular component (CC) and molecular function (MF).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/e8d44b45f7b4d58e9968d4aa.png"},{"id":77854773,"identity":"8683bf41-8451-477d-a1ee-748b4e74bdee","added_by":"auto","created_at":"2025-03-06 07:34:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":426750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide annotation of the locus of the co-location between LncRNA and mRNA\u003c/strong\u003e. Gene Ontology (GO) analysis of the genes that are co-located with the long non-coding RNAs (lncRNAs) around 100 Kb in the sheep genome which are considered to be regulated by those lncRNAs.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/7ac483e31cc21cd3586fdb66.png"},{"id":77855873,"identity":"33c49484-4b5a-40e8-9794-5ea914f230e4","added_by":"auto","created_at":"2025-03-06 07:42:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":647934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide annotation of the significant expression genes\u003c/strong\u003e. The results of the KEGG analysis demonstrate the pathways involved in the co-location of differential long non-coding (lnc)RNAs to target genes. These pathways are perhaps related to tail lipid metabolism, as illustrated by the red.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/5316bb15252b87313b3cce57.png"},{"id":77854777,"identity":"0c1d6226-1f23-4cee-86e9-91f2ea2214f3","added_by":"auto","created_at":"2025-03-06 07:34:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":398345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel miRNA distribution and association signatures\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e Novel miRNAs structures reveled in this study. \u003cstrong\u003eB\u003c/strong\u003e The line graph is organized according to the relative expression level values of miRNAs. Each subplot features a grey line, representing the line graph of the relative expression of miRNAs in a cluster under varying experimental conditions. The blue line signifies the average of the relative expression of all miRNAs in the cluster under different experimental conditions, while the red line serves as the reference point. The upper line indicates up-regulation and the lower line indicates down-regulation. The x-axis denotes the experimental conditions, whilst the y-axis indicates the relative expression. \u003cstrong\u003eC\u003c/strong\u003e Heatmap shows the cluster analysis of differential expressed miRNA, and red indicates high expression and blue indicates low expression.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/92851c2b9c31c4eed9923873.png"},{"id":92391959,"identity":"1ce06e4e-dc60-4887-9f02-96c2d796ca90","added_by":"auto","created_at":"2025-09-29 08:46:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4398513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/9f302596-b461-40ec-8404-87d17be6e516.pdf"},{"id":77854774,"identity":"6511e604-c709-456f-acae-0a7f4d702399","added_by":"auto","created_at":"2025-03-06 07:34:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2614103,"visible":true,"origin":"","legend":"","description":"","filename":"S1.mRNAgene.FPKM.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/e2a30991830cb0590ab9dc73.xlsx"},{"id":77855875,"identity":"91dff408-dada-440a-b6b7-f25f0a8dd3d6","added_by":"auto","created_at":"2025-03-06 07:42:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4895664,"visible":true,"origin":"","legend":"","description":"","filename":"S2.DEGs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/d46d06e05bbeeb72d75a21c3.xlsx"},{"id":77856021,"identity":"816d93ed-27fc-44ab-aad7-d862890e486a","added_by":"auto","created_at":"2025-03-06 07:50:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":707714,"visible":true,"origin":"","legend":"","description":"","filename":"S3.lncRNAmRNAcolocationGOresults.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/4f61e52a94c1613f080af5bc.xlsx"},{"id":77854780,"identity":"4db21301-1359-4751-8379-630bc7bd90bd","added_by":"auto","created_at":"2025-03-06 07:34:01","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":113528,"visible":true,"origin":"","legend":"","description":"","filename":"S4.DEGsKEGGresults.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/8472cf1d2883165ac401d63c.xlsx"},{"id":77856022,"identity":"bc82e446-df38-4b92-bc62-72b9bc7523ae","added_by":"auto","created_at":"2025-03-06 07:50:01","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14223,"visible":true,"origin":"","legend":"","description":"","filename":"S5.miRNADEresults.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6146587/v1/acb5a15e0d39f141c8752f03.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFats are mainly composed of triacylglycerols (TAGs), which function as the primary storage medium for energy in animals [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In circumstances where nutrients are plentiful, excess nutrients are transferred to fat reserves, which are then stored in the liquid phase of fat [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These fat reserves provide essential sustenance during periods of food scarcity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. One of the most effective strategies for reducing energy expenditure is the reduction of fat, a process which has a substantial impact on the quality of meat produced by livestock [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA significant disparity exists in the capacity of various sheep breeds to deposit fat in the tail [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In light of these findings, sheep can be categorised into two distinct groups: fat-tailed sheep, which include breeds such as the Kazakh and Altay sheep, and thin-tailed sheep, which include the Chinese Merino and Suffolk sheep, among others [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The fat-tailed trait in sheep, specifically the rump, is a biological trait that is essential for survival in challenging environments and has played a pivotal role in specific historical periods [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The fat-tailed trait of northern Chinese sheep is the result of long-term evolution and selection under complex and harsh natural environmental conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These conditions have endowed the breeds with strong adaptability to the local severe natural ecological environment, including excellent characteristics such as tolerance to coarse feed, strong stress resistance, rapid growth and development, and high meat and fat production performance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Some of them evolve a strategy in sheep, which is deposit the fat into their tails with unknown reasons [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The Kazakh sheep (HUS) is named after its round and fat tail, which is full of fat, and its main characteristics are coarse wool, big tail, and both meat and fat [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. On the contrary, tail fat deposition capacity of prolific Suffolk sheep (SFK) differs greatly between fat-tailed and lean-tailed sheep, but the genetic characteristics and molecular mechanisms of fat deposition are still unclear.\u003c/p\u003e \u003cp\u003ePrevious studies had explored the genetic mechanism of the fat tail formation by whole-genome sequencing [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Molecular markers such as SNP and CNV, are used to define some candidate genes which related to the phenotypes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Meanwhile, a set of gene locus are detected by the obvious discrepancy of allele frequency. It is reported that a set of RNAs, such as miRNAs and lncRNAs, play an important role in regulating the fat deposition [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Wang, \u003cem\u003eet al\u003c/em\u003e. found the fat deposition related gene \u003cem\u003ePDGFD\u003c/em\u003e has alternative splicing function, led to relevant with tail fat deposit [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Transcription regulator \u003cem\u003eIRF2BP2\u003c/em\u003e also represented allele differential expression in muscle tissue, resulting a vital role in muscle growth process [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, we are trying to explore the mechanism of fat deposit with transcriptome sequencing. We bred the F2 generation of prolific Suffolk and Kazakh sheep, and extract the caudal fat tissue for sequencing samples. A cluster of RNAs have been detected that participate in the metabolic pathways, which impact the adipogenesis and tail lipid formation. We also defined a group of miRNAs and lncRNAs which are differential expressed in fat tissue. Those findings provide an opportunity for us to further look inside in the caudal fat formation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003ch2\u003eSample preparation\u003c/h2\u003e\n\u003cp\u003eThree healthy adult Kazakh sheep (HUS) which is fat-tailed, F2 generation (F2) of crossbreeding between Kazakh (HUS) and Suffolk sheep (SFK) and adult Suffolk sheep, lean-tailed, of nearly the same age were selected and uniformly reared and managed. Tail tissue samples are collected in December when tail fat deposition is at its highest. Tail fat samples were collected in the shortest possible time using specially treated surgical instruments for RNA experiments (all from the same side), frozen in liquid nitrogen and stored in a refrigerator at -80℃ for back-up.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eRNA isolation\u003c/h2\u003e\n\u003cp\u003eRNA was extracted from tail tissues, and the samples underwent stringent quality control based on the following criteria. First, agarose gel electrophoresis was used to assess RNA integrity and detect potential DNA contamination. Second, RNA concentration and purity were preliminarily quantified. Finally, the integrity of RNA was accurately measured.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eLibrary preparation\u003c/h2\u003e\n\u003cp\u003eFor library construction, strand-specific libraries were prepared using a ribosomal RNA (rRNA) depletion method. Total RNA was first treated to remove rRNA, and the remaining RNA was fragmented into short segments of 250\u0026ndash;300 bp. These fragments served as templates for first-strand cDNA synthesis using random oligonucleotide primers. The second-strand cDNA was then synthesized using dNTPs as substrates. The double-stranded cDNA was purified, end-repaired, A-tailed, and ligated with sequencing adapters. The second strand of cDNA was degraded using the USER enzyme, and the remaining cDNA was amplified by PCR to obtain the final strand-specific library. To ensure library quality, a Qubit fluorometer was used for preliminary quantification, diluting the library to 1 ng/\u0026micro;L. The Agilent 2100 Bioanalyzer was then employed to assess the insert fragment size, which was expected to range from 250 to 300 bp. After confirming the expected insert size, qPCR was conducted for precise quantification of the library\u0026apos;s effective concentration, which was required to exceed 2 nM to ensure high-quality samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, libraries that passed quality control were pooled based on their effective concentration and sequencing data requirements for Illumina PE150 sequencing. In paired-end sequencing (PE150), each end of the cDNA insert is sequenced to a length of 150 bp, with the insert fragment serving as the sequencing unit. Paired-end sequencing not only provides sequence information from both ends of the insert fragment but also offers insights into the length between the two ends, facilitating downstream assembly and alignment.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eRNA sequencing\u003c/h2\u003e\n\u003cp\u003eThe sequencing was performed using the Sequencing by Synthesis (SBS) method. In this approach, the flow cell was loaded with four fluorescently labeled dNTPs, DNA polymerase, and adapter primers. During each cycle of extension, a fluorescently labeled dNTP was incorporated into the complementary DNA strand, releasing a fluorescence signal specific to the incorporated nucleotide. These signals were captured by the sequencer, and computational software converted the fluorescence data into sequencing peaks, providing the nucleotide sequence of the target fragments.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData analysis\u003c/h2\u003e\n\u003cp\u003eRNA-seq data is applied quality control and adapter trimming by Trim Galore v0.6.4_dev and mapped to the reference genome using STAR v2.7.2d. And we performed reads summarization for genomic features via featureCounts v2.0.0. The raw read counts normalization and differential expression were determined by DESeq2 v1.34.0. The number of reads matched to an expressed gene was standardized as fragments per kilobase of exon per million mapped fragments (FPKM) values. The genes that exhibited |log2(fold change)| \u0026ge;\u0026thinsp;2 and adjusted P\u0026thinsp;\u0026le;\u0026thinsp;0.05 in the comparisons between fat-tailed/fat-rumped and thin-tailed individuals were considered as differentially expressed genes. GO enrichment analysis used the enrichGO function provided within the R package clusterProfiler v4.2.2.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eF2 generation of the thin- and fat-tailed sheep\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSheep tail fat deposition are economically important traits and have been hot research topics in recent years [17]. Tail fat can satisfy the early human demand for lipids, and with the improvement of living standards, the demand for high-fat meat decreases [15]. Therefore, it is of great significance to explore the important genes regulating tail fat deposition and muscle growth rate in sheep to breed low-fat and high-yield meat sheep. In order to identify the key regulator of fat deposition, a breeding program was initiated. This involved the generation of F2 offspring from thin-tailed Suffolk sheep and fat-tailed Kazakh sheep (Fig. 1D). The Kazakh sheep is known as fat-tailed (Fig. 1A-B), whereas the Suffolk sheep have thin-tailed phenotype (Fig. 1C). The caudal fat tissue of the Kazakh and F2 was extracted, and it was found that the Kazakh tissue was larger than that of the F2 tail (Fig. 1E). However, the regulatory mechanism is still unclear.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis reveals potential regulatory modules\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo define the reconcile capability, we utilized the RNA sequencing to monitor gene expression dynamics using the Kazakh, Suffolk and F2 tail fat tissue. The FPKM distribution were calculated and showed an average level (Fig. 2A, Supplementary Table 1). Then, we compared the gene expression values and detected the significant up-regulated and down-regulated genes (Fig. 2B, Supplementary Table 2), such as \u003cem\u003ePDGFD\u003c/em\u003e and \u003cem\u003eIRF2BP2\u0026nbsp;\u003c/em\u003ewhich are consistent of previous studies. There are 1,049 up-regulated genes and 451 down-regulated genes in Kazakh sheep (HUS) than the F2 generation (CSH). A comparison was made between HUS and SFK with 754 genes that are expressed at high levels and 711 that are expressed at low levels.\u0026nbsp;SFK has 830 up-regulated expressed genes compared to CSH, and 602 down-regulated genes. As previously mentioned, the identification of potential regulatory genes can be achieved through the analysis of the set of genes exhibiting differential expression levels in fat tails in comparison to thin tails (Fig. 2C). To further identify regulatory genes, we overlapped the differential expression genes. We compared each of the three groups, HUS, SFK and CSH, and further narrowed it down based on the overlap of differentially expressed genes (Fig. 3A-D), 93 genes are defined as potential key effectors in lipid metabolism. The Pearson correlation reflects the linear relationship among those three groups, which prove the robust (Fig. 3E). To specify the fat-tail regulated genes, we overlapped the high expressed genes in HUS and SFK, which compared with CSH (Fig. 3F). There are 36 genes up-regulated in HUS and SFK, and GO enrichment analysis results showed 5 core genes (\u003cem\u003eBDH1, EPHX1, BCAT2, FASN, ACACA\u003c/em\u003e) participate in lipid metabolic process and fatty acid biosynthetic process (Fig. 3H-G). Based on previous analysis, we could conclude that the high-throughput sequencing data making it suitable for analyzing the regulatory modules with biological differences across groups and we defined 5 candidate genes could play an important role in mediating the formation of fat tail.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional signature analysis of coding and non-coding RNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecognizing that function of non-coding RNAs is common challenge in RNA-seq data. For the past few decades, lncRNA have been regarded as transcripts of minimal biological significance [2, 16]. Nevertheless, there is mounting evidence to suggest that lncRNA is widespread in eukaryotes and plays a crucial role that regulated gene expression, particularly in mammals [11, 28]. Here, we attempt to explore the mechanism of fat tail formation by Gene Ontology (GO) analysis of the differential expressed lncRNA and around mRNA which are co-located in genome (Fig. 4A-C, Supplementary Table 3). First, we analyzed the mRNAs which may regulated by highly-expressed lncRNA in HUS than SFK mainly enriched in response to stimulus or stress, immune system process and cell-cell adhesion including protein binding function (Fig. 4A). By comparing the HUS with CSH, those genes participated in response to stress, extracellular region and receptor binding (Fig. 4B). Through the result of SFK and CSH, response to stimulus or stress and cytokine receptor binding represent the signal transduction pathways could be the potential executive processes (Fig. 4C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further identify the functional elements involved in tail fat deposition, differential expressed genes (DEGs) were annotated to multiple pathways, which help us to find the unique program modulating the fat deposition in sheep tail. KEGG results shows that DEGs are enriched in Metabolic pathways, TCA cycle, Fatty acid metabolism, Fat digestion and absorption, Fatty acid biosynthesis (Fig. 5, Supplementary Table 4). These results revealed the significant genes controls the fat deposit in tail by participating fat-related pathways, and offered us an opportunity to declare the principle of fat deposition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovel miRNA identification\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmall RNA (sRNA) idiosyncratically recognized and bound the RNA-induced silencing complex (RISC) and inhibited gene expression [29, 30]. sRNAs play an important role in regulating almost all events at the cellular level, such as individual development, cell proliferation and differentiation, tumor development and antiviral resistance, and are important members of the highly complex RNA regulatory network in cells, especially miRNA [22, 31]. To gain further insight into whether miRNA have any functional role in fat deposition, we evaluated a set of novel miRNAs and divide them into four subclusters according to the its relative expression levels (Fig. 6A-B, Supplementary Table 5). Finally, we examined the total differential expressed miRNAs and defined up-regulated miRNAs (\u003cem\u003enovel_401, novel_72, novel_120, novel_440, novel_85, novel_171, oar-miR-218a, novel_74, novel_144, oar-miR3958-3p, novel_107, novel_51, novel_46, oar-miR-543-3p\u003c/em\u003e) in fat-tailed samples (Fig. 6C). These findings underline the miRNAs perhaps assume a vital role in fat tail deposition as epigenomic regulators.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, our RNA-seq dataset provides a unique platform to investigate sheep specific genes in fat deposition of tail, identifying regulatory modules, such as protein-coding genes, lncRNAs and miRNAs. Although most of these miRNAs are novel detected and those function remains to be further investigated. In summary, our study shows that mRNA could be regulated by lncRNAs in cold environment which is response to stress and be part of lipid metabolism pathways leading fat tail. And miRNAs as epigenomic regulator controls the fat-deposit-regulated genes expression level in fat tissue, to which leads the deposition in tail.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to explore the genes regulating tail fat deposition and muscle growth rate in sheep, with the ultimate goal of breeding low-fat and high-yield meat sheep. The findings from the F2 generation of thin-and fat-tailed sheep crosses, along with transcriptomic, functional signature, and miRNA analyses, have provided valuable insights into the complex molecular mechanisms underlying tail fat deposition.\u003c/p\u003e \u003cp\u003eThe transcriptomic analysis using RNA sequencing of tail fat tissues from Kazakh, Suffolk, and F2 sheep was a crucial step. The identification of differentially expressed genes (DEGs) between these groups has shed light on potential regulatory genes. The large number of up-regulated and down-regulated genes in Kazakh sheep compared to the F2 generation, as well as between other pairwise comparisons, indicates significant genetic differences contributing to tail fat deposition.\u003c/p\u003e \u003cp\u003eThe overlapping of DEGs across the three groups (HUS, SFK, and CSH) to define 93 potential key effectors in lipid metabolism is a significant finding. These genes are likely to be central to the regulatory network controlling fat deposition in the sheep tail. The Pearson correlation among the groups validates the reliability of the high-throughput sequencing data, ensuring that the identified regulatory modules are robust and biologically relevant. The genes such as \u003cem\u003ePDGFD\u003c/em\u003e and \u003cem\u003eIRF2BP2\u003c/em\u003e, which were consistent with previous studies, further support the validity of our approach and the potential importance of these genes in fat deposition.\u003c/p\u003e \u003cp\u003eThe exploration of the functions of non-coding RNAs, especially lncRNAs, through GO analysis, has revealed new aspects of fat tail formation. The finding that mRNAs potentially regulated by highly-expressed lncRNAs in Kazakh sheep compared to Suffolk sheep are enriched in processes such as response to stimulus or stress, immune system processes, and cell-cell adhesion is intriguing. This suggests that these biological processes may be indirectly related to fat deposition, perhaps through their effects on the overall physiological state of the tissue.\u003c/p\u003e \u003cp\u003eThe GO analysis comparing different groups also showed that genes were involved in various processes related to stress response and receptor binding. These results imply that the regulation of fat deposition in the sheep tail is a complex process, with multiple biological pathways and molecular functions interacting. The enrichment of DEGs in metabolic pathways, TCA cycle, fatty acid metabolism, fat digestion and absorption, and fatty acid biosynthesis as revealed by KEGG analysis further emphasizes the role of these canonical metabolic pathways in tail fat deposition. Identifying these pathways provides a framework for understanding how genes control fat deposition in the tail and offers potential targets for genetic manipulation in breeding programs.\u003c/p\u003e \u003cp\u003eThe identification of novel miRNAs and their classification into sub-clusters based on relative expression levels is an important addition to our understanding of fat deposition regulation. The up-regulated miRNAs in fat-tailed samples, such as \u003cem\u003enovel_401, novel_72, and oar-miR-218a\u003c/em\u003e, suggest that these miRNAs may act as epigenomic regulators in fat tail deposition. miRNAs are known to play crucial roles in post-transcriptional gene regulation, and their involvement in fat deposition indicates a complex regulatory network at the miRNA level.\u003c/p\u003e \u003cp\u003eAlthough most of these miRNAs are newly detected and their functions remain to be fully investigated, our results provide a starting point for future research. Understanding how these miRNAs interact with other regulatory elements, such as lncRNAs and protein-coding genes, will be essential for a comprehensive understanding of the molecular mechanisms of fat deposition in sheep tails.\u003c/p\u003e \u003cp\u003eOverall, our study has integrated multiple levels of molecular analysis to identify potential regulatory elements in sheep tail fat deposition. The identification of coding genes, lncRNAs, and miRNAs involved in this process provides a rich resource for further research. Future studies could focus on validating the functions of the identified genes and miRNAs through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. For example, gene knockout or overexpression studies in sheep or relevant cell models could be used to directly assess the impact of these genes on fat deposition.\u003c/p\u003e \u003cp\u003eFurthermore, understanding the interactions between different regulatory elements, such as how lncRNAs regulate mRNAs and how miRNAs fine-tune the expression of fat-deposition-related genes, will be crucial [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This knowledge could be applied to \u003cb\u003edevelop\u003c/b\u003e new breeding strategies to manipulate tail fat deposition, aiming to produce sheep with lower fat content in the tail while maintaining high-quality meat production. In conclusion, our study has laid a solid foundation for future research in this area, with potential implications for the sheep breeding industry.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the following grants: The Science and Technology Innovation Talents Project of Corp (2023CB007-03), The National Nature Science Foundation of China (31660651), China Agriculture Research System (CARS-39-07), Young Science and Technology Top Talent Program of Tianshan Talent Training Program in Xinjiang Province (2022TSYCCX0124), Xinjiang Agriculture Research System (XJARS-09-26), Project of Corps Science and Technology in Key Areas (2024AB017).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eL.G. performed the majority of the experiments, collected and analyzed the data, and drafted the initial manuscript. Y.Z. and J.Y. assisted in experimental work, data analysis, and interpretation, and helped in drafting the manuscript. Z.Z. conducted statistical analyses and prepared data visualizations. Y.L. and J.W. contributed to sample collection and conducted laboratory experiments. L.W. and P.W. contributed to the study design, provided essential resources, and offered critical feedback during manuscript preparation. Z.Z. conceived and designed the study, supervised the overall project, and provided critical revisions to the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eRNA-seq data have been deposited in the GEO database (GEO: GSE290403) and are publicly available as of publication. This study does not report original algorithms. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e\n\u003ch2\u003eEthics Statement\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eEthics Committee Approval: This study was approved by the Ethics Committee (Approval Number: A2024-008). \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSahu, B., et al., \u003cem\u003eRole of Distinct Fat Depots in Metabolic Regulation and Pathological Implications.\u003c/em\u003e Rev Physiol Biochem Pharmacol, 2023. \u003cstrong\u003e186\u003c/strong\u003e: p. 135-176.\u003c/li\u003e\n\u003cli\u003eBakhtiarizadeh, M.R. and S.A. 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Feldman, \u003cem\u003eWhite adipocytes in subcutaneous fat depots require KLF15 for maintenance in preclinical models.\u003c/em\u003e J Clin Invest, 2024. \u003cstrong\u003e134\u003c/strong\u003e(13).\u003c/li\u003e\n\u003cli\u003eFei, X., et al., \u003cem\u003eTranscriptome reveals key microRNAs involved in fat deposition between different tail sheep breeds.\u003c/em\u003e PLoS One, 2022. \u003cstrong\u003e17\u003c/strong\u003e(3): p. e0264804.\u003c/li\u003e\n\u003cli\u003eHan, J., et al., \u003cem\u003eQuantitative proteomic analysis identified differentially expressed proteins with tail/rump fat deposition in Chinese thin- and fat-tailed lambs.\u003c/em\u003e PLoS One, 2021. \u003cstrong\u003e16\u003c/strong\u003e(2): p. e0246279.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6146587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6146587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTail fat in sheep (\u003cem\u003eOvis aries\u003c/em\u003e), has evolved mainly in response to cold weather for better energy storage. As things stand, too much tail fat in sheep can lead to a reduction in feed utilisation and is also unpopular with consumers due to the excessive fat content in the tail of sheep. Therefore, the need to find the mechanism of tail fat formation is obvious. In this study, we elected to utilise Kazakh sheep, prolific Suffolk sheep, and their hybrid F2 generation as research objects. Sheep transcriptome sequencing technology was employed to screen and explore target candidate genes related to sheep tail fat deposition. Comparison with RNA-seq data from fat-tailed and thin-tailed tissue, the LncRNA-mRNA-miRNA axis was identified as main functional pathway in the formation of fat in tail. Our results offer valuable insights into the fat deposition of sheep and provide a significant genomic resource for future genetic studies and the enhancement of genome-assisted breeding in sheep and other domestic animals.\u003c/p\u003e","manuscriptTitle":"Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-06 07:33:56","doi":"10.21203/rs.3.rs-6146587/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":"fd5036f7-3af8-4579-b09d-6475a426f249","owner":[],"postedDate":"March 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T08:41:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-06 07:33:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6146587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6146587","identity":"rs-6146587","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}