www.nature.com/scientificreports OPEN Tissue‑specifc regulatory mechanism of LncRNAs and methylation in sheep adipose and muscle induced by Allium mongolicum Regel extracts Jiangdong Xue1,2,4, Qi Lv1,3,4, Erdene Khas1, Chen Bai1, Bingjie Ma3, Wangjiao Li3, Qina Cao1, Zejun Fan1 & Changjin Ao1* Allium mongolicum Regel (A. mongolicum) is a perennial and xerophytic Liliaceous allium plant in high altitude desert steppe and desert areas. Feeding A. mongolicum greatly reduced unpleasant mutton favor and improves meat quality of sheep. We analyzed epigenetic regulatory mechanisms of water extracts of A. mongolicum (WEA) on sheep muscle and adipose using RNA‑Seq and whole‑genome Bisulfte sequencing. Feeding WEA reduced diferentially expressed genes and long non‑coding RNAs (lncRNAs) between two tissues but increased diferentially methylation regions (DMRs). LncRNA and DMR targets were both involved in ATP binding, ubiquitin, protein kinase binding, regulation of cell proliferation, and related signaling pathways, but not unsaturated fatty acids metabolism. Besides, tissue specifc targets were involved in distinct functional annotations, e.g., Golgi membrane and endoplasmic reticulum for muscle lncRNA, oxidative phosphorylation metabolism for adipose lncRNA, dsRNA binding for muscle DMRs. Epigenetic regulatory networks were also discovered to discovered essential co‑regulated modules, e.g., co‑regulated insulin secretion module (PDPK1, ATP1A2, CACNA1S and CAMK2D) in adipose. The results indicated that WEA induced distinct epigenetic regulation on muscle and adipose to diminish transcriptome diferences between tissues, which highlights biological functions of A. mongolicum, tissue similarity and specifcity, as well as regulatory mechanism of mutton odor. Sheep (Ovis aries) is a typical domestic animal, with a farming history more than 8000 years1,2. Along with the movements of nomadic societies, sheep have spread almost globally by a “transportation hub” in eastern Eurasia, Mongolian Plateau region3,4. Tis type of ruminants has played an important role in human society, and is a major source of meat, milk, and fber in the form of wool5–7. As a unique and necessary food source for the human diet, mutton has abundant nutrients, such as iron, zinc, selenium, fatty acids and vitamins 8. Mutton consumption in north China (e.g., in Inner Mongolia), is more popular than that in other regions, because meat favor and odor is more pleasant than that in other regions, especially for older sheep 9. Mutton odor originates from two types of branched chain fatty acids (BCFAs): 4-methyl nonanoic acid (MNA) and 4-ethyloctanoic acid (EOA) present in all the adipose tissue; and the 3-methylindole (MI), skatole or indole originated from pastoral diets. Researchers have been focusing on meat quality of sheep adipose and muscle 10. For instance, storage of uncured mutton adipose tissue samples under anoxic conditions limits lipid oxidation but enhances mutton odor intensity11. Sheep natural mutations that afect muscle growth (e.g., callipyge, CLPG) have potential to signifcantly improve lamb-meat quality 12. In sheep muscle, some motifs are mutated and bind to corresponding transcriptional factors to regulate promoter activity and musclin gene expression 13. Dietary improves nutritional 1Inner Mongolia Key Laboratory of Animal Nutrition and Feed Science, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China. 2College of Animal Science and Technology, Inner Mongolia University for Nationalities, Tongliao 028000, China. 3Key Laboratory of Animal Genetics, Breeding and Reproduction in Inner Mongolia Autonomous Region, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China. 4These authors contributed equally: Jiangdong Xue and Qi Lv. *email: [email protected] Scientifc Reports | (2021) 11:9186 | https://doi.org/10.1038/s41598-021-88444-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ value and meat quality of mutton, e.g., dietary vitamin E increases polyunsaturated fatty acid (PUFA) content and decreases saturated fatty acid (SFA) content of mutton14. Allium mongolicum Regel (A. mongolicum, also known as Mongolia leek) is a type of perennial and xerophytic Liliaceous allium plant in high altitude desert steppe and desert areas and is considered as one specifc food source for pleasant meat favor15,16. Tis plant is involved in multiple biological and physiological processes. For instance, polysaccharides extracted from A. mongolicum afect signaling molecules of sheep peripheral blood lymphocytes in vitro by regulating the concentrations of Ca 2+, NO, cAMP and cGMP 17. A. mongolicum and its extracts increase the average daily gain (ADG), feed intake and feed remuneration of broiler chickens through growth-related hormones18,19. However, the underlying regulatory mechanism of how this plant efectively elimi- nates mutton odor in sheep is still unknown. Epigenetics are heritable molecular determinants of phenotype by adjusting gene expression without hav- ing changes in DNA sequences. Epigenetic mechanisms are implicated during development in utero and at the cellular level, and bioactive food components may trigger protective epigenetic modifcations throughout life 20. Epigenetic features include DNA methylation, long non-coding RNAs (lncRNAs), histone modifcations, micro- RNA expression, and chromatin structure, which regulate gene expression through distinct mechanisms21,22. For instance, DNA methylation represses gene expressions by interfering the normal function of transcriptional activators on promoters or the repeated sequences across gene bodies 23,24; while lncRNAs infuence expression or stability of protein-coding RNAs in a localized, gene-specifc fashion or by targeting large chromosomal regions25,26. One characteristic of these epigenetic markers is tissue specifcity, e.g., muscle and adipose tissues. Tere is evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may have subsequent efects on disorder of adipose later in life 27,28. Specifcally, DNA methylation plays an important role in several diferentiation processes and possibly in adipocyte diferentiation, e.g., WGEF expres- sion regulates adipogenesis through DNA methylation change in 3T3-L1 cells29. Besides, muscle development is also regulated with epigenetic modifcation. Genes important for myogenesis are up regulated by histone acetyltransferases and other chromatin modifers correlated with several transcription factors, including Pax7, MyoD and Mef230. In addition, 1407 diferentially expressed lncRNAs that showed consistent expression patterns between skeletal muscles of Large White pigs and Mashen pigs, indicating the involvement of lncRNAs in muscle development31. Moreover, phosphotyrosine phosphatase inhibitor bisperoxovanadium resets the cell fate program by specifc epigenetic regulations, which leads to an enhanced regenerative potential of muscle stem cell 32. Tere- fore, understanding epigenetic mechanism of feeds is essential for muscle and adipose in animal production. In this study, we applied whole-genome Bisulfte sequencing and RNA-sequencing to analyze epigenetic changes in adipose and muscle induced by water extracts of A. mongolicum (WEA). We systematically investi- gated alterations of transcriptome and epigenome and studied potential epigenetic mechanism by target genes regulated by lncRNAs and methylation, which highlights signifcance of A. mongolicum as a unique sheep feed to improve meat quality. Results High quality of transcriptome sequencing data. We obtained 15.08G ± 1.23G clean reads afer qual- ity control, and quality scores of over 93.24% reads were more than Q30. Maximum error rate was less than 0.08%, indicating high quality of sequencing data (Table S1, Fig. 1a). Over 81.05% clean reads were mapped to genome, including over 68.45% uniquely mapping (Table S2). Reads were mainly mapped to protein coding regions (73.55% ± 2.88%) and lncRNAs (0.72% ± 0.12%) (Fig. 1b). 324,356 transcripts were assembled by String- Tie, including 43,674 protein coding transcripts. Remaining transcripts were analyzed with exon number, tran- script length, known annotation, expression level and protein coding potential (see methods). Finally, 15,222 transcripts with non-coding potential were predicted by CPC, CNCI and PfamScan, and defned as the lncRNAs (Fig. 1c). Tese lncRNAs were classifed into intronic lncRNAs (55.8%), intergenic lncRNAs (34.4%) and anti- sense lncRNAs (9.8%) (Fig. 1d). Compared with mRNA, lncRNAs had less exon numbers, shorter sequence lengths and open reading frame (ORF) lengths (Fig. 1e, Figure S1a, S1b). Distributions of overall mRNA expres- sions were consistent among 12 samples (Figure S1c). In addition, the expressions of diferent treatments of the same tissues were of high correlation coefcient (0.877 ± 0.011), higher than that between diferent tissues (0.670 ± 0.022) (Figure S2). Expression of lncRNAs was lower than mRNA expression (Fig. 1f). Methylation characteristics distinguished by methylation context. Average Bisulfte sequencing depth of bases was 17.70 ± 0.49, while sequencing coverages of bases (sequencing depth > 10 ×) were 66.42– 78.82% (Table S3). Totally we obtained 42.56 Gb C sites and average coverage depth was 5.03 ± 0.20 (Table S4). Base numbers were 267.98 ± 12.69 (Mb), 1309.16 ± 45.64 (Mb) and 3870.56 ± 148.42 (Mb) for CG, CHG and CHH respectively (Table S4). Average methylation level of CG was 70.60%