Characterization And Analysis Of Differently Expressed MicroRNAs In The Ovaries Of Uniparous And Multiple Goats (Capra Hircus)

Anhui White goat which is higher in the reproductive capacity than other goat, it is the most important productive property of people in the wide plain and hills between Wanbei and Jianghuai area. Economic efficiency of a flock of goats is dependent on its total productivity, and the productivity is more dependent on fertility and prolificacy of the female goats than any other components.However, hircine fecundity is relatively low, and the trait is difficult to improvement by conventional breeding methods for its low heritability which ranged 0.09 to 0.14. Therefore, researchers pin their hope on molecular assisted breeding technology for improving goat’s fecundity rapidly. Ovaries are one of the most important reproductive organs of animals, as they play a very important role in reproductive regulation, and its ovulation rate is high relative with goats’s kidding rate. MicroRNAs (miRNAs) are a group of small non-coding RNAs that can modulate gene expression by inhibiting mRNA translation or regulating mRNA degradation at the post-transcriptional level.Many studies indicated that miRNAs play critical roles in almost all ovarian biological processes, including folliculogenesis, follicle development, follicle atresia, luteal development and regression.For understanding the role of miRNAs in kidding rate regulation of goat and also may help to identify miRNAs which could be potentially used to increase hircine ovulation rate and kidding rate in the future. In this study, we sequenced the small RNAs in the ovarian tissues of multiple and uniparous Anhui White goats during follicular phase by Illumina Solexa technology, then analyzed the differentially expressed miRNAs, predicted novel miRNAs, and made GO enrichment and KEGG pathway analysis of target genes in two miRNA libraries. The main results are indicated as follows:1. In order to identify differentially expressed miRNA during follicular phase in the ovaries of multiple and uniparous Anhui White goats, two small RNA libraries were constructed by Solexa sequencing. A total of 12,000,000 raw reads were obtained. After quality disposing,5,948,837 and 5,945,145 clean reads which obtained from multiple and uniparous goats remained for further analysis. Subsequently, all identical sequence reads were classified as groups, and 160,284 and 235,735 unique sequences were obtained. The majority of the small RNAs were 20-24 nt range. Sequences 22 nt in length, the typical size of miRNA, peaked at length distribution, respectively accounted for 56.93% and 54.26% of the total sequence reads in the multiple and uniparous libraries.2. We aligned the clean reads to the miRNA precursor/mature miRNAs of all known animals in the miRBase 20.0 database. The sequence and count of families (no specific species) were obtained which could be found in the two libraries. MiRNA expression is more concentrated and total expression levels of the top 20 miRNAs account for more than 80% of the total expression levels of all miRNAs in each library. And, let-7b is the highest expressed miRNA in the two libraries.3. After normalized the expression level and difference comparison,1317 and 1441 conserved miRNAs were identified in the multiple and uniparous libraries. Among them, 1008 miRNAs were co-expressed,309 and 433 miRNAs were specifically expressed in the multiple and uniparous libraries, respectively. The overwhelming majority of these specifically expressed miRNAs’expression level were very low (under 5), whereas the expression level of 4 and 9 specific expressed miRNAs in multiple and uniparous libraries were higher than 1000. The highest specific expressed miRNA in multiple library was miR-29c which reached counts of 5,214, and the highest specific expressed miRNA in uniparous library was miR-6406 which reached counts of 42,571.4. GO annotation and KEGG Pathway analyses were implemented on target genes of all miRNA in two libraries. Results indicate that In GO analysis, miRNA targets were significantly enriched to different terms in two libraries, and the significantly enriched terms in uniparous library contained the terms in multiple library. As for KEGG pathway analysis, it is worth to note that Focal adhesion, ABC transporters, Carbohydrate digestion and absorption, Peroxisome, Starch and sucrose metabolism, and Progesterone-mediated oocyte maturation were involved in the significantly enriched pathway in the ovaries during follicular phase of multiple goats. These significantly enriched pathways may imply that the organism was coping to the criteria of follicular phase in multiple goats.5. RT-PCR was applied to detect the expression level of 5 (miR-378a, miR-10a, miR-202-5p, miR-84a, and let-7d-5p, and 3 of them were differentially expressed) randomly selected miRNAs in multiple and uniparous hircine ovaries, and the results were consistent with the Solexa sequencing data.