Transcriptome analysis of the pectoral muscles of local chickens and commercial broilers using Ribo-Zero ribonucleic acid sequencing

<div><p>Background</p><p>The molecular mechanisms underlying meat quality and muscle growth are not clear. The meat quality and growth rates of local chickens and commercial broilers are very different. The Ribo-Zero RNA-Seq technology is an effective means of analyzing transcript groups to clarify molecular mechanisms. The aim of this study was to provide a reference for studies of the differences in the meat quality and growth of different breeds of chickens.</p><p>Results</p><p>Ribo-Zero RNA-Seq technology was used to analyze the pectoral muscle transcriptomes of Gushi chickens and AA broilers. Compared with AA broilers, 1649 genes with annotated information were significantly differentially expressed (736 upregulated and 913 downregulated) in Gushi chickens with Q≤0.05 (Q is the P-value corrected by multiple assumptions test) at a fold change ≥2 or ≤0.5. In addition, 2540 novel significantly differentially expressed (SDE) genes (1405 upregulated and 1135 downregulated) were discovered. The results showed that the main signal transduction pathways that differed between Gushi chickens and AA broilers were related to amino acid metabolism. Amino acids are important for protein synthesis, and they regulate key metabolic pathways to improve the growth, development and reproduction of organisms.</p><p>Conclusion</p><p>This study showed that differentially expressed genes in the pectoral tissues of Gushi chickens and AA broilers were related to fat metabolism, which affects meat. Additionally, a large number of novel genes were found that may be involved in fat metabolism and thus may affect the formation of meat, which requires further study. The results of this study provide a reference for further studies of the molecular mechanisms of meat formation.</p></div

Schematic representation of the alternative splicing events in the samples.

<p>The y-value represents the number of observed splicing events, and the x-value shows the major six alternative splicing types in Gushi chickens and AA chickens, respectively. RI = retained introns; ES = exon skipping; A3SS = alternative 3' splicing sites; A5SS = alternative 5' splicing sites; MEX = mutually exclusive exons.</p

Characteristics of the reads from pectoral libraries obtained from 2 breeds of chicken.

<p>Characteristics of the reads from pectoral libraries obtained from 2 breeds of chicken.</p

Venn diagram of global annotated genes, transcript isoforms and novel genes expressed in Gushi chickens and AA chickens by Ribo-Zero RNA sequencing.

<p>Venn diagrams show the distribution of annotated genes (A), transcript isoforms (B), and novel genes (C) detected in Gushi chickens and AA broilers. Red indicates Gushi chickens, and blue indicates AA chickens.</p

Genome coverage map.

<p>The outermost circle is the genome, the first inner circle shows the chromosome coverage of the AA chicken (AA), and the second shows that of the Gushi chicken (Gushi). The number on the outer circle represents the chromosome number, the number on the inner circle represents the position on the chromosome, and the unit is Mb.</p

Validation of RNA-Seq data by qRT-PCR.

<p>Data were analyzed by the 2^–ΔΔCt method using GAPDH as a reference gene. The results are presented as fold changes in expression. Each column represents the means ± SEM from 3 biological replicates with each measurement repeated 3 times. Different lowercase letters indicate significant differences in expression levels between the two breeds (P ≤ 0.05). Black bars = qRT-PCR; Gray bars = FPKM from RNA-Seq.</p

Distribution of the mapped reads on different regions of the chicken reference genome.

<p>Noncoding regions include all the 5′UTR, 3′UTR and other noncoding RNA regions. UTR = untranslated region.</p

GO analyses of differentially expressed genes in Gushi chickens and AA chickens.

<p>A shows the GO function classification (level 2), and B shows the 30 most significantly enriched GO terms.</p

KEGG pathway analyses of differentially expressed genes in Gushi chickens and AA chickens.

<p>A shows the KEGG pathway classification, and B shows the 30 most significantly enriched KEGG pathways.</p

Comparison of differentially expressed genes between the two breeds.

<p>Scatter plot showing the correlation of gene abundance. Red points represent genes upregulated by at least two fold at FDR ≤ 0.05, blue points represent genes downregulated at the same thresholds, and grey dots indicate transcripts that did not change significantly.</p