Analysis of alternative splicing events by RNA sequencing in the ovaries of Xiang pig at estrous and diestrous

Alternative splicing (AS) is an important mechanism for controlling gene expression, which regulates multiple biological processes in higher organisms. Chinese indigenous Xiang pig has distinctive biological characteristics, such as small size, early sexual maturity, lower litter size and not very clear exhibition of estrous behaviors. To further understand how AS responds to estrous cycles in Xiang pig, the genome-wide analysis of AS events was performed by RNA-seq method in Xiang pig ovaries at diestrous and estrous. Using ASprofile program, we analyzed twelve basic AS events in Xiang pig ovaries and identified 68,775 AS events in 15,142 genes from diestrous ovaries and 69,493 AS events in 15,291 genes from the estrous ovaries with average 4.54 splicing events. 94.4-95.5% of expressed genes underwent alternative splicing in this tissues. The frequencies of AS events were similar to each other at diestrous and estrous. Transcription start site (TSS) was the predominant type of AS events, followed by transcription terminal site (TTS), and skipped exon (SKIP). The remaining type of AS events, e.g., intron retention (IR) and alternative exon ends (AE), showed the lower frequencies. Further comparison analysis of gene expression indicated that 4,433 genes had at least one splice variant differentially expressed during estrous, whereas only 2,382 of them were differentially expressed at gene level. Numerous genes involved in gonad development and hormone metabolism were differentially regulated through AS. Twelve genes with different types of alternatively splicing were validated by using RT-PCR method. The GO annotation and KEGG pathway analysis clearly revealed that a lot of DEGs (differentially expressed genes) and DSGs (differentially spliced genes) were involved in follicular development and ovarian steroid biosynthesis. A large number of DSGs, although not differentially expressed, were enriched in circadian rhythm and several signaling pathways. These pathways potentially regulated the female animal reproductive function in gene and/or AS level. Our results suggested that alternative splicing play an essential role in regulation of gene expression in female pigs during estrous. Numerous genes involved in gonad development, hormone metabolism, circadian rhythm were differentially regulated through alternative splicing.