Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos

Abstract

Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our understanding of the location-specific and breed-specific genes and pathways but also provides some candidate molecular targets for increasing muscle products and meat quality by genetic control.

Fig 1. Skeletal muscle variations in leg muscle (LM) and pectoral muscle (PM) between Heiwu duck (H) and Peking duck (P) during development.

A. The increase in the weight of four skeletal muscles from post-hatching week 1 (W1) to week 16 (W14). B. The changing rate of skeletal muscle weight to carcass weight from post-hatching week 1 (W1) to week 16 (W14). C. The variation in the weight of the four skeletal muscles in embryo from embryonic day 15 (E15) to day 28 (E28). D. The changing rate of skeletal muscle weight to carcass weight from embryonic day 15 (E15) to day 28 (E28). At each time point (W1, W4, W10, etc), statistically significant differences are indicated by different letters (a, b, c, etc). At a given time point, any samples that are not significantly different are labeled with the same letter.

Fig 2. The distribution of DEGs in each library.

A: The numbers of differentially expressed genes in each comparison. The up-regulated genes are shown in red, whereas the down-regulated genes are shown in green. “A” was the control group, and “B” was the experimental group in “A vs B”. B. The distribution of DEGs that were found to be commonly and specifically expressed between locations in Experiment 1. C. The distribution of DEGs that were found to be commonly and specifically expressed between breeds in Experiment 2.

Fig 3. RT-PCR validates the correction of RNA-Seq.

The relative expression levels were calculated using β-actin and GAPDH as the internal controls. RPKM: the number of reads per kilo bases per million reads (RPKM).

Fig 4. Histogram presents gene ontology classification.

The left axis indicates the percentage of the specific category of genes in the main category. The right axis indicates the number of genes in a category. A, B, C and D represent the H-PM vs H-LM.GO, P-PM vs P-LM.GO, P-LM vs H-LM.GO, and P-PM vs H-PM.GO, respectively.