Identification and Comparative Analysis of Long Non-Coding RNA in the Skeletal Muscle of Two Dezhou Donkey Strains

Abstract

Long non-coding RNA (lncRNA) has been extensively studied in many livestock. However, compared with other livestock breeds, there is less research regarding donkey lncRNA function. It has been reported that lncRNA plays an important role in the timing control of development, aging, and death of livestock. Therefore, the study of donkey skeletal muscle lncRNA is of great significance for exploring donkey meat production performance. In this study, RNA-Seq was used to perform high-throughput sequencing of skeletal muscle (longissimus dorsi and gluteus) of two Dezhou donkey strains (SanFen and WuTou). The differentially expressed lncRNAs were screened between different strains and tissues. Then candidate genes for conjoint analysis were screened based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Finally, the accuracy of the sequencing data was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Herein, we identified 3869 novel lncRNAs and 73 differentially expressed lncRNAs. Through the comparison between groups, the specific expression of lncRNAs were found in different strains and muscle tissues. Importantly, we constructed the lncRNA-miRNA-mRNA interaction network and found three important candidate lncRNAs (MSTRG.9787.1, MSTRG.3144.1, and MSTRG.9886.1) and four candidate genes (ACTN1, CDON, FMOD, and BMPR1B). Analysis of the KEGG pathway indicates that the TGF-β signaling pathway plays a pivotal role in the growth and development of skeletal muscle in Dezhou donkey strains.

Keywords: Dezhou donkey; TGF-β signaling pathway; lncRNA; skeletal muscle.

Figure 1

Boxplot of the novel lncRNAs prediction score (a): Novel lncRNAs prediction score wih CNCI software (Version 2, GitHub, San Francisco, CA, USA). (b): Novel lncRNAs prediction score wih CPC software (Version 2, CBI, USA).

Figure 2

Different class codes of expression level of skeletal muscle lncRNAs. potentially novel isoform (fragment): at least one splice junction is shared with a reference transcript; i: a transfrag falling entirely within a reference intron; o: generic exonic overlap with a reference transcript; u: unknown, intergenic transcript; x: exonic overlap with reference on the opposite strand; WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle and SanFen donkey gluteus muscle, respectively.

Figure 3

The Characteristics Comparison of Transcripts (a): Distribution of exon number for mRNAs and lncRNAs. (b): Expression level and a number of mRNAs and lncRNAs. (c): Distribution of lncRNA ORFs length. (d): Distribution of mRNA ORFs length.

Figure 3

The Characteristics Comparison of Transcripts (a): Distribution of exon number for mRNAs and lncRNAs. (b): Expression level and a number of mRNAs and lncRNAs. (c): Distribution of lncRNA ORFs length. (d): Distribution of mRNA ORFs length.

Figure 4

lncRNAs bar chart and venn diagram. (a): Differentially expressed lncRNAs in different groups. (b): All expressed lncRNAs venn diagram among the four groups. (c): Differentially expressed lncRNAs venn diagram among the four groups (WB vs. SB, WT vs. ST, WB vs. WT, SB vs. ST). WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle, and SanFen donkey gluteus muscle, respectively.

Figure 5

RT-qPCR results of lncRNAs with significant differences in WB vs. SB and WT vs. ST comparison group. The figure shows the quantitative results of RNA-seq and RT-qPCR; the line graph shows the FPKM value of the sequencing and the histogram shows the relative quantitative results; the significance test: * p ≤ 0.05, ** p ≤ 0.01. WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle and SanFen donkey gluteus muscle, respectively.

Figure 6

The ceRNA network. Green squares represent lncRNAs, yellow triangles represent miRNAs, and red circles represent mRNAs. (A): WB vs. SB. (B): WT vs. ST. WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle and SanFen donkey gluteus muscle, respectively.

Figure 7

GO enrichment analysis of the ceRNA network. (a): WB vs. SB. (b): WT vs. ST. WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle, and SanFen donkey gluteus muscle, respectively.

Figure 8

Significantly enriched KEGG terms of the ceRNA network. (a): WB vs. SB. (b): WT vs. ST. Y-axis: pathway name; X-axis: enrichment factor; Bubble size reflects the number of differential expressions enriched to a certain pathway/all quantities in the background; Color reflects the significance of differential expression in a certain process. WB, WT, SB, and ST represent WuTou donkey longissimus dorsi muscle, WuTou donkey gluteus muscle, SanFen donkey longissimus dorsi muscle, and SanFen donkey gluteus muscle, respectively.

Figure 9

TGF-β signaling pathway. Red indicates up-regulated genes and blue indicates down-regulated genes.