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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.

29 30 2 1 2 Abstract 3 Background/Aims: Litter size is one of the most important reproductive traits in pig breeding, which is affected by 4 multiple genes and the environment. Ovaries are the most important reproductive organs and have a profound impact 5 on the reproduction efficiency. Therefore, genetic differences in the ovaries may contribute to the observed 6 differences in litter size. Although QTLs and candidate genes have been reported to affect the litter size in many pig 7 breeds, however, the findings cannot elucidate the marked differences of the reproductive traits between breeds. The 8 aim of present work is to elucidate the mechanisms of the differences for the reproductive traits and identify 9 candidate genes associated with litter size in Xiang pig breed. Methods: The changes in ovary transcriptome and 10 alternative splicing were investigated at estrus between Xiang pigs with large and small litter size by RNA-seq 11 technology. The RNA-seq results were confirmed by RT-qPCR method. Results: We detected 16,219 -16,285 expressed 12 genes and 12 types of alternative splicing (AS) events in Xiang pig samples. A total of 762 differentially expressed genes 13 were identified by XL (Xiang pig group with larger litter size) vs XS (Xiang pig group with small litter size) sample 14 comparisons. A total of 34 genes were upregulated and 728 genes were downregulated in XL ovary samples compared 15 with the XS samples. Alternative splicing (AS) rates in XL samples were slightly lower than that observed in XS samples. 16 Most of differentially expressed genes were differentially regulated on AS level. Eleven candidate genes were 17 potentially identified to be related to Xiang pig fecundity and litter size, which may be closely related to the gonad 18 development, oocyte maturation or embryo quality. Conclusion: The significant changes in the expression of the 19 protein-coding genes and the level of alternative splicing in estrus ovarian transcriptome between XL and XS groups 20 probably are the molecular mechanisms of phenotypic variation in litter size. 21 22

The biological clock has been studied to play a critical role in the reproductive system of various living organisms like swine. To examine the effects of estrus cycle on the expression of ovarian biological clock-related gene in Xiang pig, in this study, we analyzed the expression and alternative splicing of biological clock-related genes in ovary during estrus and diestrus periods. In total, we detected 90 clock-related genes expressed in the ovaries of the Xiang pigs and found 33 clock-related genes differentially expressed between estrous and diestrous stages. We identified 44 differential splicing events from the transcripts of 34 biological clock-related genes. Furthermore, we also found 20 genes including the core clock components, arntl and cry1 were differentially regulated only at AS level and 14 genes, including per1 and clock, were differentially regulated at both expression and AS levels. We also proved that the core clock genes per1, cry1, clock and arntl and the clock-related genes, ppp1cb and ntrk1 were rhythmically expressed in Xiang pig ovaries by RT-qPCR experiments. The results demonstrated that the biological clock in the ovaries of Xiang pigs might play an important role in regulating the ovarian physiological functions by the transcriptional and post-transcriptional regulation.