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Background Although lots of quantitative trait loci (QTLs) and genes present roles in litter size of some breeds, the information might not make it clear for the huge diversity of reproductive capability in pig breeds. To elucidate the inherent mechanisms of heterogeneity of reproductive capability in litter size of Xiang pig, we performed transcriptome analysis for the expression profile in ovaries using RNA-seq method. Results We identified 1,419 up-regulated and 1,376 down-regulated genes in Xiang pigs with large litter size. Among them, 1,010 differentially expressed genes (DEGs) were differently spliced between two groups with large or small litter sizes. Based on GO and KEGG analysis, numerous members of genes were gathered in ovarian steroidogenesis, steroid biosynthesis, oocyte maturation and reproduction processes. Conclusions Combined with gene biological function, twelve genes were found out that might be related with the reproductive capability of Xiang pig, of which, eleven genes were recognized as hub genes. These genes may play a role in promoting litter size by elevating steroid and peptide hormones supply through the ovary and facilitating the processes of ovulation and in vivo fertilization.

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.

Background/AimsLitter size is one of the most important reproductive traits in pig breeding, which is affected by multiple genes and the environment. Ovaries are the most important reproductive organs and have a profound impact on the reproduction efficiency. Therefore, genetic differences in the ovaries may contribute to the observed differences in litter size. Although QTLs and candidate genes have been reported to affect the litter size in many pig breeds, however, the findings cannot elucidate the marked differences of the reproductive traits between breeds. The aim of present work is to elucidate the mechanisms of the differences for the reproductive traits and identify candidate genes associated with litter size in Xiang pig breed.MethodsThe changes in ovary transcriptome and alternative splicing were investigated at estrus between Xiang pigs with large and small litter size by RNA-seq technology. The RNA-seq results were confirmed by RT-qPCR method.ResultsWe detected 16,219 - 16,285 expressed genes and 12 types of alternative splicing (AS) events in Xiang pig samples. A total of 762 differentially expressed genes were identified by XL (Xiang pig group with larger litter size) vs XS (Xiang pig group with small litter size) sample comparisons. A total of 34 genes were upregulated and 728 genes were downregulated in XL ovary samples compared with the XS samples. Alternative splicing (AS) rates in XL samples were slightly lower than that observed in XS samples. Most of differentially expressed genes were differentially regulated on AS level. Eleven candidate genes were potentially identified to be related to Xiang pig fecundity and litter size, which may be closely related to the gonad development, oocyte maturation or embryo quality.ConclusionThe significant changes in the expression of the protein-coding genes and the level of alternative splicing in estrus ovarian transcriptome between XL and XS groups probably are the molecular mechanisms of phenotypic variation in litter size.