Transcriptome sequencing data
A total of 1,438,500,450 raw reads were obtained by RNA-seq high-throughput sequencing of tail fat from Bashby sheep and F2 sheep. A total of 1,338,966,230 clean reads were obtained after filtering the original data obtained by sequencing, it accounts for 90.64~95.61% of high-quality sequence data, and Q20% is more than 99% in all samples, and GC% is between 47~49.50%, as shown in Table 1. The reliability of the sequencing results can be used for subsequent data analysis.
Sequence alignment results with reference genomes
The comparison between the transcriptome data and the reference gene sequence was shown in Table 2. The comparison efficiency between the original sequence data and the reference genome of each sample ranged from 85.28 to 91.06%.
Screening and clustering analysis of differentially expressed lncRNAs
Analyze the differential expression of samples by Deseq2 software and determine the differentially expressed lncRNAs in tail fat of different breeds of sheep by Volcano map, and calculate whether the difference was significant (Figure 1). The results showed that 270 lncRNAs were downregulated and 458 lncRNAs were upregulated. Cluster analysis was then performed on the differentially expressed lncRNAs screened out from the tail fat of different breeds of sheep (Figure 2) to determine the expression patterns of DElncRNAs in the tail fat of different tail types of sheep.
Prediction of lncRNA target genes
We used the co-localization of lncRNA and protein-coding genes to predict the target genes of lncRNA, to further study the function of the lncRNA obtained from ovine tail fat, in which the upstream and downstream 100 kb of lncRNA was used as the threshold value of co-localization. The results showed that 728 DElncRNAs targeted 17,850 target genes. In these predictions, one lncRNA targeted multiple mRNAs, and simultaneously, multiple lncRNAs targeted the same mRNA. MSTRG.36913, MSTRG.24995, MSTRG.37980, MSTRG.38164, MSTRG.36912, MSTRG.8169 and MSTRG.31389 target genes related to fat deposition and fat metabolism, such as THRSP, FASN, SCD and GPAM, etc. Partial results of lncRNA targeting genes involved in fat deposition and fat metabolism are shown in Table 3.
Functional and pathway analysis of DElncRNAs targets
GO enrichment was performed by DElncRNAs (Figure 3). The integral component of the membrane, positive regulation of GTPase activity, inflammatory response, immune response, lateral plasma membrane, defense response to the virus were significantly enriched in several terms, and the integral component of the membrane was most significantly enriched. DElncRNAs were analyzed by KEGG pathway analysis (Figure 4). It is mainly enriched in Viral carcinogenesis, Type I diabetes mellitus, T cell receptor signaling pathway, Systemic lupus erythematosus, Staphylococcus aureus infection, Primary immunodeficiency, Osteoclast differentiation, Natural killer cell mediated cytotoxicity, Measles, Leishmaniosis, Intestinal immune network for IgA production, Hematopoietic cell lineage, Graft-versus-host disease, Fc gamma R-mediated phagocytosis, Cytokine-cytokine receptor interaction, Chemokine signaling pathway, B cell receptor signaling pathway, Autoimmune thyroid disease, Allograft rejection, Alcoholism. In addition, a total of 7 of 728 DElncRNAs (MSTRG.37980, MSTRG.38164, MSTRG.36912, MSTRG.36913, MSTRG.8169, MSTRG.24995 and MSTRG.31389) and the target genes (SCD, GPAM, THRSP, FASN, NDUFC2, WDTC1 and PIK3R1) related to fat metabolism were screened by investigating literatures.
Quantitative analysis verified sequencing accuracy
To verify the accuracy of RNA-seq data, 7 significantly DElncRNAs (MSTRG.37980, MSTRG.38164, MSTRG.36912, MSTRG.36913, MSTRG.8169, MSTRG.24995 and MSTRG.31389) related to fat deposition were screened to be analyzed by qRT-PCR. The results (Figure 5) showed that the quantitative and RNA-seq results of 7 lncRNA in the tail adipose tissue of Bashby and F2 were the same, indicating that the RNA-seq results were more accurate and reliable, indicating that expressed assessment and identification of lncRNAs were persuasive and could be used for subsequent experiments.
Differential expression of lncRNAs in different tissues
In order to explore the DElncRNAs in different tissues of Bashby sheep (CG) and F2 (TG), we conducted relative quantitative analysis of 7 DElncRNAs by qPCR (Figure 6). We found that there were differences and similarities in the expression patterns of DElncRNAs in tissues of Bashby and F2. The significant expression level of tail fat in F2 was MSTRG.37980, MSTRG.38161, MSTRG.24995, while that in Bashby was only MSTRG.37980 and MSTRG.24995. MSTRG.37980 and MSTRG.38164 were highly expressed of the large intestine in F2, but lower in Bashby. This indicated that the expression of DElncRNAs in different tissues was different among species.
Difference analysis of DElncRNAs in the same tissue of different tail fat type sheep
The expression levels of 7 DElncRNAs were determined in 11 parts of Bashby (CG) and F2 (TG) (Figure 7.). We can know that these 7 DElncRNAs are extremely significant or significantly different in the tail fat tissues of Bashby and F2, and the expression of MSTRG.37980, MSTRG.38166, MSTRG.36913 and MSTRG.24995 in F2 is lower than that of Bashby, while the expression of MSTRG.36912, MSTRG.8169 and MSTRG.31389 is higher in F2 (P<0.05). For subcutaneous fat tissue, the expression of MSTRG.37980, MSTRG.38164, MSTRG.36913, and MSTRG.36912 in F2 was significantly lower than that in Bashby, while the expression of MSTRG.8169 was opposite (P<0.05). Meanwhile, the expression abundance of MSTRG.37980 in longissimus dorsi muscle, MSTRG.37980 and MSTRG.24995 in small intestine, MSTRG.38164 and MSTRG.36913 in liver of F2 sheep was significantly lower than that of Bashby (P<0.05). And the expression abundance of MSTRG.36913 in quadriceps femoris, MSTRG.24995 in rumen, MSTRG.36912 and MSTRG.36389 in arm triceps of F2 sheep was significantly higher than that of Bashby (P<0.05). These DElncRNAs not only differ in tail fat tissues, but also indirectly regulate fat deposition in other tissues.
Expression maps of DElncRNAs target genes in different tissues of sheep with different tail types
The target gene expression levels determined at 11 sites in Bashby sheep (CG) and F2 (TG) are basically the same as lncRNAs, except for the PIK3R1 in the longissimus dorsi muscle (Figure 8). We could see that the expression levels of SCD, THRSP and FSAN in F2 tail fat tissue were significantly lower than those of Bashby, while NDUFC2, WDTC1 and PIK3R1 were significantly higher in Bashby (P<0.05). Meanwhile, the expression abundance of GPAM in liver, THRSP in subcutaneous fat and liver, FSAN in small intestine, NDUFC2 in subcutaneous fat and duodenum of F2 sheep was significantly lower than that of Bashby (P<0.05). And the expression abundance of SCD in cecum, THRSP in longissimus dorsi muscle, FASN in rumen, DOUFC2 in arm triceps, liver, rumen, and small intestine, PIK3R1 in longissimus dorsi muscle, arm triceps, quadriceps femoris, liver, rumen, large and small intestine of F2 sheep was significantly higher than that of Bashby (P<0.05). This indicates that the target genes are differentially expressed in different tissues of sheep with different tail types, thereby indirectly or directly participating in ovine tail fat deposition.