Transcriptome Profiling Identifies Differentially Expressed Genes in Huoyan Goose Ovaries between the Laying Period and Ceased Period

<div><p>The Huoyan goose is famous for its high egg-laying performance and is listed as a nationally protected domestic animal by the Chinese government. To elucidate the key regulatory genes involved in Huoyan goose egg laying, RNA from ovarian tissue during the ceased and laying periods was sequenced using the Illumina HiSeq 2000 sequencing platform. More than 12 million reads were produced in ceased and laying libraries that included 11,896,423 and 12,534,799 clean reads, respectively. More than 20% of the reads were matched to the reference genome, and 23% of the reads were matched to reference genes. Genes with a false discovery rate (FDR) ≤0.001 and log₂ratio ≧1 or ≤−1 were characterized as differentially expressed, and 344 up-regulated and 344 down-regulated genes were classified into functional categories. Twelve genes that are mainly involved in pathways for reproduction regulation, such as steroid hormone biosynthesis, GnRH signaling pathways, oocyte meiosis, progesterone-mediated oocyte maturation, steroid biosynthesis, calcium signaling pathways, and G-protein coupled receptor signaling pathway were selected for validation by a quantitative real-time polymerase chain reaction (qRT-PCR) analysis, the qRT-PCR results are consistent with the general expression patterns of those genes from the Illumina sequencing. These data provide comprehensive gene expression information at the transcriptional level that might increase our understanding of the Huoyan goose's reproductive biology.</p></div

Validation of the gene expression profile by qRT-PCR.

<p>Total RNA extracted from the ovarian tissues that was measured by RT-PCR analysis; relative expression levels were calculated according to the 2^−ΔΔCT method using beta-actin as an internal reference gene and the ceased period group as the calibrator (relative expression  = 1). Error bar shows the standard deviation. Gene symbols indicating different genes refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113211#pone-0113211-t002" target="_blank">Table 2</a>. *<i>P</i><0.05, **<i>P</i><0.01.</p

Comparison of gene expression levels between the ceased period and laying period libraries.

<p>DEGs were filtered using FDR ≤0.001 and |log₂ratio| ≧1 as a threshold. Red spots represent up-regulated genes, and green spots indicate down-regulated genes. Blue spots represents genes that did not show obvious changes between the ceased period and laying period samples.</p

Evaluation of the expression profile variation between RNA-Seq and qRT-PCR for selected genes.

<p>Evaluation of the expression profile variation between RNA-Seq and qRT-PCR for selected genes.</p

Distribution of gene coverage identification for differentially expressed genes.

<p>Gene coverage is used to assess the depth of DGE sequencing and refers to the percentage mapped by reads of each gene.</p

Distribution statistics of reads mapped to reference genes.

<p>The horizontal axis represents the relative read position, which is calculated as the ratio between a read's location on the gene and the gene length, and the vertical axis represents the numbers of reads that are mapped to genes.</p

Alignment statistical results of the ceased_period library and laying_period library.

<p>Alignment statistical results of the ceased_period library and laying_period library.</p

Putative candidate genes of the DEGs associated with goose laying.

<p>Putative candidate genes of the DEGs associated with goose laying.</p

Different components of the raw reads in each sample.

<p>The percentages of reads containing N, adaptors, clean reads and low-quality reads. The numbers in parentheses indicate the percentage of each type of read among the total raw reads.</p

Additional file 1: Figure S1. of Molecular cloning and expression analysis of adiponectin and its receptors (AdipoR1 and AdipoR2) in the hypothalamus of the Huoyan goose during different stages of the egg-laying cycle

Nucleotide and deduced amino acid sequences of adiponectin. The nucleotide (black) and deduced amino acid (blue) sequences are shown and numbered on the left. The nucleotide sequence is numbered from the 5’ end. The first methionine (M) is the first deduced amino acid. The C1Q domain (amino acids 105–241) is shaded. The start codons (ATG) and the stop codons (TAA) are marked in bold red. (TIFF 315 kb