Transcriptome sequencing reveals key potential long non-coding RNAs related to duration of fertility trait in the uterovaginal junction of egg-laying hens

Abstract

Duration of fertility, (DF) is an important functional trait in poultry production and lncRNAs have emerged as important regulators of various process including fertility. In this study we applied a genome-guided strategy to reconstruct the uterovaginal junction (UVJ) transcriptome of 14 egg-laying birds with long- and short-DF (n = 7); and sought to uncover key lncRNAs related to duration of fertility traits by RNA-sequencing technology. Examination of RNA-seq data revealed a total of 9977 lncRNAs including 2576 novel lncRNAs. Differential expression (DE) analysis of lncRNA identified 223 lncRNAs differentially expressed between the two groups. DE-lncRNA target genes prediction uncovered over 200 lncRNA target genes and functional enrichment tests predict a potential function of DE-lncRNAs. Gene ontology classification and pathway analysis revealed 8 DE-lncRNAs, with the majority of their target genes enriched in biological functions such as reproductive structure development, developmental process involved in reproduction, response to cytokine, carbohydrate binding, chromatin organization, and immune pathways. Differential expression of lncRNAs and target genes were confirmed by qPCR. Together, these results significantly expand the utility of the UVJ transcriptome and our analysis identification of key lncRNAs and their target genes regulating DF will form the baseline for understanding the molecular functions of lncRNAs regulating DF.

Figure 1

Duration of fertility trait in hens. (a,b) Histogram illustrates the distribution of DF trait within the population of egg-laying hens (n = 304). DN: the days post-insemination until the last fertile and FN: the numbers of fertile eggs laid after artificial insemination (AI). Bell-shaped indicate the normal distribution and the density curve was determined by standard normal distribution parameters N (µ, σ) where µ is the mean and σ is the standard deviation.

Figure 2

The panel of a tissue section of the UVJ of egg-laying hen. (a,b) long-DF hens and (c,d) short-DF hens. A rounded-like shape known as sperm storage tubule (SST) was more embedded in the UVJs of long-DF hens compared with short-DF hens. Data are presented as mean ± SEM (n = 4). P < 0.01 (Student’s t-test). The red arrow indicates the SSTs, Scale bar = 100 μm.

Figure 3

Classification of sequencing raw reads from (a) Long-DF and (b) Short-DF libraries in hens.

Figure 4

The distribution of transcript length, exon number and expression level of lncRNAs and mRNAs identified in UVJ samples of long-DF and short-DF hens. (a) Distribution of identified lncRNA and mRNA transcript lengths. (b) Distribution of exon numbers of lncRNA and mRNA transcripts. (c) Expression distribution of the lncRNA and mRNA transcripts.

Figure 5

The differentially expressed (DE) lncRNAs between the long- and short-DF hens. The red and blue dots represent up- and down-regulated lncRNAs respectively. The grey dots represent the lncRNAs without significant differential expression.

Figure 6

The regulatory network of the 8 differentially expressed lncRNAs involved in regulating the duration of fertility trait in egg-laying hens was constructed. Yellow and green nodes represent upregulated and downregulated lncRNAs in the long-DF group respectively. Pink nodes represent the predicted lncRNA target genes. Lines represent the regulatory relationships between lncRNAs and their potential target genes.

Figure 7

The 30 most enriched functional classifications of DE-lncRNAs target genes between long and short-DF hens. (a) The Gene Ontology (GO) of the DE-lncRNAs target genes. The ontology covers three categories: Biological Process, Cellular Component, and Molecular Function. (b) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway regulated by DE-lncRNAs target genes of long and short-DF hens.

Figure 8

Validation of differentially expressed lncRNAs by qPCR. (a,b) Fold change expression of down-regulated lncRNAs in long DF hens compared with the short DF hens; (c,d) Fold change expression of up-regulated lncRNAs in long DF hens compared with short DF hens. Data are reported for three technical replicates in all biological samples. (e–i) The expression levels of lncRNA target genes between long and short DF hens. Each histogram represents the level of the target gene relative expression level. Error bars indicate standard error of the mean (SEM). Asterisks symbolized P-value significance *p ≤ 0.05, and **p ≤ 0.01, based on assuming unequal variances Student’s t-test.