Characterization and Comparative Transcriptomic Analysis of Skeletal Muscle in Pekin Duck at Different Growth Stages Using RNA-Seq

Abstract

Skeletal muscle, accounting for approximately 50% of body weight, is the largest and most important tissue. In this study, the gene expression profiles and pathways in skeletal muscle of Pekin duck were investigated and compared at embryonic day 17, 21, and 27 and postnatally at 6 months of age. An average of 49,555,936 reads in each sample was obtained from the transcriptome libraries. Over 70.0% of alternative splicing (AS) in each sample was mainly alternative 5' first exon (transcription start site)-the first exon splicing (TSS) and alternative 3' last exon (transcription terminal site)-the last exon splicing (TTS), indicating that TSS and TTS were the most common AS event in Pekin ducks, and these AS events were closely related to the regulation of muscle development at different growth stages. The results provided a valuable genomic resource for selective breeding and functional studies of genes. A total of 299 novel genes with ≥2 exons were obtained. There were 294 to 2806 differentially expressed genes (DEGs) in each pairwise comparison of Pekin duck. Notably, 90 DEGs in breast muscle and 9 DEGs in leg muscle were co-expressed at all developmental points. DEGs were validated by qPCR analysis, which confirmed the tendency of the expression. DEGs related to muscle development were involved in biological processes such as "endodermal cell differentiation", "muscle cell cellular homeostasis", "skeletal muscle tissue growth" and "skeletal muscle cell differentiation", and were involved in pathways such as oxidative phosphorylation, ECM-receptor (extracellular matrix receptor) interaction, focal adhesion, carbon metabolism, and biosynthesis of amino acids. Some DEGs, including MYL4, IGF2BP1, CSRP3, SPP1 and KLHL31, as well as LAMB2, LAMA2, ITGB1 and OPN, played crucial roles in muscle growth and development. This study provides valuable information about the expression profile of mRNAs and pathways from duck skeletal muscle at different growth stages, and further functional study of these mRNAs and pathways could provide new ideas for studying the molecular networks of growth and development in duck skeletal muscle.

Keywords: DEG; Pekin duck; pathway; skeletal muscle; transcriptome.

Figure 1

Annotation and classification of SNPs (single nucleotide polymorphisms) and InDels (insertion-deletions) in Pekin duck. (A) SNPs of breast muscle; (B) SNPs of leg muscle; (C) InDels of breast muscle; (D) InDels of leg muscle.

Figure 2

The predicted number of alternative splicing in Pekin ducks during different incubation stages. (A–D) represent the predicted number of alternative splicing in E17d, E21d, E27d and M6, respectively.

Figure 3

Number of DEGs during skeletal muscle development in Pekin duck.

Figure 4

Venn diagram of differentially expressed genes among the three comparison groups. (A) The co-expressed DEGs in breast muscle of Pekin duck; (B) the co-expressed DEGs in leg muscle of Pekin duck.

Figure 5

GO enrichment analysis of DEGs in Pekin duck. (A) PE17B_vs_PE21B; (B) PE21B_vs_PE27B; (C) PE27B_vs_PM6B; (D) PE17L_vs_PE21L; (E) PE21L_vs_PE27L; (F) PE27L_vs_PM6L. Note: The abscissa were GO terms, the ordinate on the left was percentage of genes in all genes annotated with GO, on the right was the number of genes.

Figure 6

KEGG annotation of DEGs in Pekin duck. (A) PE17B_vs_PE21B; (B) PE21B_vs_PE27B; (C) PE27B_vs_PM6B; (D) PE17L_vs_PE21L; (E) PE21L_vs_PE27L; (F) PE27L_vs_PM6L. Note: Each circle represented a KEGG pathway, the name of which was shown on the left legend. Abscissa indicated enrichment factors, showing the proportion of (a) to (b), (a) was the ratio of differentially expressed genes in the pathway with all DEGs in all pathways, (b) was the ratio of genes in the pathway with all genes in all pathways. The bigger the Rich factor is, the more significant the pathway is. The color of circle represented q value which is adjusted p value by multiple hypothesis testing. Thus, the smaller the q value is, the more significant the pathway is; the circle size represented the number of differentially expressed genes annotated with the pathway, the bigger circle size is, the higher number of genes is.

Figure 7

qPCR verification of DEGs. “*” was considered significant difference (p < 0.05); “**” was considered highly significant difference (p < 0.01).