Hybrid Sequencing in Different Types of Goat Skeletal Muscles Reveals Genes Regulating Muscle Development and Meat Quality

Abstract

Domestic goats are commonly reared for meat and milk production in several regions of the world. However, the genetic mechanism underlying muscle development and meat quality of goats is limited. Therefore, the aim of this study was to identify known and novel genes regulating muscle development and meat quality of goats using second- and third-generation sequencing technologies. To achieve this, the meat quality and transcriptomes of longissimus dorsi (LD) and biceps femoris (BF) muscle tissues of Lingqiu Greyback goats were examined and compared. Differentially expressed genes (DEGs) and isoforms (DEIs) were functionally annotated. Results showed that 45,574 full-length transcripts covering 18,491 loci were characterized, and 12,566 genes were co-expressed in all samples. Differential expression analysis identified 231 DEGs, including 45 novel genes in the LD and BF muscles of the goats. Additionally, 1173 DEIs were found, in which 642 novel isoforms were identified in this study. Functional annotation and pathway analysis of the DEGs and DEIs revealed that some of them were associated with muscle growth and lipid metabolism. Overall, the findings of this study contribute to the understanding of the transcriptomic diversity underlying meat quality and muscle development of goat.

Keywords: full-length transcriptome; goat; growth; hybrid sequencing; meat quality; skeletal muscle.

Figure 1

Full-length transcriptomic sequencing improves the understanding for goat transcriptome. (A) Hybrid sequencing strategy used in this study. PacBio third generation and Illumina second generation sequencing were used to obtain the full-length and expression level of transcript, respectively. (B) Comparison between goat RefSeq from NCBI and PacBio data obtained from this study. (C) Classification of full-length transcripts obtained from third generation sequencing. (D) Known transcripts (XM_*) of TNNT1 already annotated by NCBI and newly discovered transcripts (NC_*) in this study. (E) Types of alternative splicing identified from full-length sequencing. ES, exon skipping; AA, alternative acceptor; AD, alternative donor; IR, intron retention.

Figure 2

Short-read sequencing quantifies the expression of genes and transcripts of goat muscles. (A) Correlations between eight samples used for short-read sequencing. The correlation between each pair of samples in the sequencing libraries was calculated from the FPKM value. Samples with a Pearson’s correlation value ≥ 0.92 were considered as the same experimental replicates. The deeper blue color represents closer correlation between samples. (B) MA plot indicating differentially expressed genes between the BF and LD muscles. The red and blue dots represent the upregulated and downregulated genes in BF compared to LD muscles, respectively. |log2(FoldChange)| > 1 and padj < 0.05 is considered up- or downregulated. (C) MA plot indicating the differentially expressed isoforms between the BF and LD muscles. (D) Cluster enrichment of differentially expressed transcripts from LD and BF samples. The gradient color key represents the adjusted p-value, which is more significantly different when having more red color. (E) RNA-seq results are consistent with qPCR data. qPCR was used to verify the expression profiles of 13 randomly selected differentially expressed genes in RNA-seq results. The gene expression levels obtained from qPCR were normalized to GAPDH and are shown as mean ± standard error of mean (SEM) (n = 4). Data from RNA-seq is shown as mean FPKM ± SEM (n = 4).

Figure 3

Gene Ontology (GO) enrichment of differentially expressed isoforms. Hyper-geometric distribution method is used. FDR ≤ 0.05 is considered as significantly enriched.

Figure 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed isoforms. Hyper-geometric distribution method is used. FDR ≤ 0.05 is considered as significantly enriched.