A global view of porcine transcriptome in three tissues from a full-sib pair with extreme phenotypes in growth and fat deposition by paired-end RNA sequencing

Abstract

Background: Elucidation of the pig transcriptome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits such as fat deposition, metabolism and growth.

Results: Here we used massive parallel high-throughput RNA sequencing to generate a high-resolution map of the porcine mRNA and miRNA transcriptome in liver, longissimus dorsi and abdominal fat from two full-sib F2 hybrid pigs with segregated phenotypes on growth, blood physiological and biochemical parameters, and fat deposition. We obtained 8,508,418-10,219,332 uniquely mapped reads that covered 78.0% of the current annotated transcripts and identified 48,045-122,931 novel transcript fragments, which constituted 17,085-29,499 novel transcriptional active regions in six tested samples. We found that about 18.8% of the annotated genes showed alternative splicing patterns, and alternative 3' splicing is the most common type of alternative splicing events in pigs. Cross-tissue comparison revealed that many transcriptional events are tissue-differential and related to important biological functions in their corresponding tissues. We also detected a total of 164 potential novel miRNAs, most of which were tissue-specifically identified. Integrated analysis of genome-wide association study and differential gene expression revealed interesting candidate genes for complex traits, such as IGF2, CYP1A1, CKM and CES1 for heart weight, hemoglobin, pork pH value and serum cholesterol, respectively.

Conclusions: This study provides a global view of the complexity of the pig transcriptome, and gives an extensive new knowledge about alternative splicing, gene boundaries and miRNAs in pigs. Integrated analysis of genome wide association study and differential gene expression allows us to find important candidate genes for porcine complex traits.

Figure 1

Comparison of the identified genes among liver, LD and AF against the UCSC database. The number in overlapped regions is the annotated genes that were expressed in all three tissues or each two tissues.

Figure 2

An example of read distributions of genes in reference genome. The schematic diagram depicts the read distributions of AY609431 (A) and AY609632 (B) on pig chromosomes 3 and 2. The red curve shows the expression level (log2 of RPKM) and the pink bar denotes the genomic regions covered by reads in RNA-seq. The yellow bar shows the transcript structures of genes in UCSC. The pink lines highlight the linkage between exons which were supported by at least two distinct junction reads. The red arrows indicate the alternative 5' splicing in panel A and the retained intron in panel B. The plot (C) shows the distribution of the number of alternative splicing events occurred in each gene and including all four types of alternative splicing models in individual 2268.

Figure 3

Discovery of miRNAs. (A) The graph shows the abundance of small RNA tags mapped to each chromosome. (B) The number of the identified mature miRNAs and putative novel miRNAs. (C) The number distribution of the putative novel miRNAs mapped to each chromosome. Abbreviation: ND, not determined.

Figure 4

The mature miRNAs ranking top 20 in normalized expression levels in each sample. AF (A), liver (B) and LD muscle (C).

Figure 5

Analyses of differentially expressed genes between individuals 2268 and 2270. The graphs of (A) to (C) show differentially expressed genes. The up-regulated genes and the down-regulated genes are respectively shown in red and green. Genes with similar expression levels are indicated in blue. The graphs of (D) to (F) indicate the GO analyses of differentially expressed genes; the bar plot represents the percentage of gene counts within each GO category. All functions or processes listed have enrichment P values < 0.05.

Figure 6

Identification of candidate genes related to phenotypes by integrated analysis of GWAS and differentially expressed genes. IGF2, CYP1A1, CKM and CES1 locate at the QTL region for heart weight, hemoglobin level at day 240, pork pH value at 9 h after slaughter and serum total cholesterol, respectively. The X-axis indicates the chromosome regions of SNPs, and the Y-axis shows the log10 radios of corrected P-value.