- To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. - The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Nutrition is one of the most important environmental factors affecting reproductive performance in sheep. - However, none of the earlier studies have involved cross-bred ewes, an excellent model for understanding the heritability of important genes.. - The F1 cross was included into the study to in- vestigate the transmission of the prolificacy phenotype into F1 generation. - ovarian mRNA and miRNA profiles was also investi- gated by maintaining approximately half of the ewes from each breed group on a flushing diet and the other half on a normal diet. - Network analysis of the differentially expressed genes and miRNAs was also implemented to reveal the gene ontology terms and pathways associated with developmental changes and reproduction in sheep.. - The aim of the present study was to investigate possible differences in the ovarian transcriptomes of two breeds and their F1 crosses. - The single-nucleotide polymorphism (SNP)-based results for the genetic rela- tionships between individuals confirmed the ancestries of the groups derived prior to the feeding experiment on the basis of the pedigree records. - On average, the Finnsheep ewes had a larger litter size of 2.7, and Texel had the lower litter size of 1.8, whereas their F1 crosses had litter sizes intermediate between those of the two breeds, with an average litter size of 2.4. - Most of the ewes were in good body condition at the start of the flushing period, with an average body condition score (BCS) of 3.0. - Any potential confounding effects of age, weight or BCS at the start of the trial were eliminated by allocating the animals between the two treatment groups (see Additional file 1: Tables S2 and S3). - The BCS of the animals that did not receive the flushing diet remained constant during the trial. - Flushing elevated blood urea concentrations in the flushing group (5.25 mmol/l) compared with the non- flushing group (4.37 mmol/l), most probably because of the rapeseed meal included in the flushing diet (Additional file 2).. - The overall quality of the mRNA-seq reads was very good, with average quality scores >. - After removal of the low- quality reads and universal Illumina adapters present in approximately 5% of the data, an average of 114.5 million reads per sample (Additional file 1: Table S4) were used for further analyses. - Altogether, 16,402 genes (baseMean ≥5) were expressed in the samples, which represented 60.6% of the known (27,054) ovine genes. - However, only 12 of the 500 top expressed genes were shared by Finnsheep and Texel ewes. - Moreover, because the ovary biopsies including follicular and connective tissues were used for RNA extraction, the heterogeneity of the tissue itself might have affected the overall gene expression dynamics. - Thus, future experiments using methods such as manual microdissection [27] may provide a clearer picture of the different cell types.. - The top ten most expressed genes were ND1 , ND2 , ND4 , ND5 , COX1 , COX2 , COIII , CYTB , ATP6 and ENSOARG which represented approximately 12% of the total clean reads. - 1 Heatmap plot of the top 20 genes with the highest genetic variance across all samples. - F1 = F1 cross of Finnsheep and Texel) of each samples in x-axis are presented at the top of the heatmap. - (total base mean of the expressed ovarian transcriptome (total base mean . - Previous studies have identi- fied mitochondrial differences in various human tissues, in which the mitochondrial transcriptome abundance is directly proportional to the energy requirements of the given tissue. - The mitochondrial transcripts in the heart have been shown to compose ~30% of the total mRNA, whereas adrenal, ovary, thyroid, prostate, testes and lung tissues make up only ~5% of the mitochondrial tran- scripts [28]. - On the basis of the differential expression analyses, we compiled a list of potential candidate genes (see Table 2 below).. - The majority of genes were upregulated in Texel because of the effect of the flushing diet. - However, most of Table 1 List of the top 20 genes with highest variance across samples. - The minor effect of the flushing diet on the litter size of Finnsheep ewes has also been previously reported in a feeding experiment 9. - Differential expression level of the genes for different comparisons are available in the additional files listed under “ Additional files (AF. - Previous studies have indicated its role in the differentiation process of the epidermis [29, 30]. - Owing to the inhibitory role of the gene, the lower level of CST6 expression most probably promotes ovulation. - Similarly, the differential expression pattern may also be associated with the dif- ference in the progression of the follicular phase of the oestrus cycle. - Because most of the genes were upregulated in the Texel group, we concluded that the flushing diet promotes developmental processes in Texel. - The majority of the GO terms were associated with heart development (angiogenesis, heart morphogenesis, heart contraction and cardiovascu- lar development). - Of the novel miRNAs, 13 were associated with chromosome 18.. - Further classification of the variants on the basis of the variant effects resulted in 23 categories with intron variants associated with 36.1% of the variants, followed by intergenic variants (20.7. - The number of SNPs was more closely related to the size of the chromosome than to the number of genes per chromosome. - None of the previously studied mutations, such as FecGH(S395F), FecTT(S427R) and FecGE(F345C), were present in any of the samples. - Unexpectedly, the SNP in one of the ewes (including two additional replicate samples) from the F1 crosses was homozygous, thus indicating that the ancestry of the animal was more than 50%. - Finnsheep (not supported by pedigree or SNP data) or that the mutation also occurred with a low frequency in the Finnish population of the Texel breed. - One explanation for this finding may be that these genes are stage-specific and may be highly expressed at some point during a later stage of the oestrus cycle.. - Only some of the Finnsheep ewes were iden- tified as carrying mutated ( HBB , n = 3) and GDF9 ( n = 5) genes, which are associated with an increased ovulation rate, thus further supporting the presence of two different lines (high and low) in Finnsheep [38]. - A close-up view of chromosome 18 revealed a peak of non-coding genes towards the 3 ′ -end, where the majority of the miRNAs were located (Additional file 1: Fig. - A large cluster of miR- NAs was identified on chromosome 18 of the sheep genome, which also contained the largest number of miRNAs. - Three genes and miRNAs each were differentially expressed between Finnsheep and Texel receiving the control diet, but none of the three genes were predicted to be targets of the three miRNAs. - From the list of 48 target genes of the eight miRNAs, only three genes, UBXN2B, PLIN5 and RASSF7, were upregulated in Finn- sheep. - However, three of the interactions that involved miR-1468, miR-433-3p, and miR-432 exhibited a clear direct relationship, and 12 of. - This prolific mu- tation did not segregate in the present sample set of Texel, but in Finnsheep, the frequency of the GDF9 c.1111G >. - Our analysis of the RNA-seq data from two globally im- portant breeds with contrasting fertility traits revealed important features regarding the ovarian transcriptome landscape. - Our findings provide strong insights into the importance of the flush- ing diet to increase sheep reproduction, particularly in breeds with low reproductive traits. - We found that Texel, a breed with a comparatively smaller litter size, was genetically responsive to the flushing diet, whereas Finnsheep were non-responsive on the basis of the ab- sence of DEGs between flushing versus the control diet (within-breed). - The identifi- cation and characterization of miRNAs that are differen- tially expressed between pure breeds should facilitate understanding of the regulatory roles of miRNAs during folliculogenesis in these breeds. - individuals but at different stages of the oestrous cycle, will allow elucidation of the roles of tissue-specific gene expression.. - The details of the feeding experiment are provided as supplementary methods (see Additional file 1).. - The oestrous cycles of the ewes were followed by pro- gesterone hormone profiling. - All blood samples (5 ml, heparinized vacuum tubes) were collected in the morning from the jugular vein at 1- to 7-day intervals from each ewe, depending on the progesterone concen- trations of the previous measurements. - To study the effects of the diets, the levels of insulin-like growth factor 1 (IGF-1), glucose, urea, albumin, and insulin were also measured during the experiment.. - Because a follow-up of the ovaries was not possible with ultrasound, the oestrous cycles were monitored by daily progesterone measurements (Additional file 2). - A sharp decrease in progesterone resulted in removal of the ovary the following day to obtain the follicular growth phase. - Immediately after removal, the ovary was washed with physiological saline, and the middle part of the ovary with blood vessels and all visible CLs were re- moved. - CLs were collected separately from the remaining ovaries of the same ewes after establishment of pregnancy (data not shown).. - To obtain an overview of the genetic relatedness of the animals and to assure the breed status, the samples were genotyped using Illumina Ovine 700 K SNP Bead- Chips (Illumina, USA). - All genotypes were called using GenomeStudio software v2011.1, and SNPs that failed to meet any of the following criteria were discarded:. - The high quality of the libraries was confirmed with an Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany), and the concentrations of the libraries were quantified via Quibit® fluorometric quanti- tation (Life Technologies, Santa Clara, CA, USA). - Additional mRNA and miRNA libraries for one of the samples from the Finnsheep flushing, Texel flushing and F1 flushing groups were sequenced. - Additionally, one of the mRNA libraries from the F1 control group was sequenced three times. - Processing of the sequencing data. - was used to trim all adapters and to perform size selection of the sequence data. - The overall RNA quantification was conducted using the CAP-miRSeq [63] tool to better understand the quality of the miRNA-seq experiment.. - The overview of the bioinformatics workflow is presented graphically in additional information 1 (Additional file 1: Fig. - To examine the possible influence of the flushing diet, we implemented differential gene expression analysis within and between breeds by incorporating diet as a second factor. - In addition, to obtain an overview of genetic differences between pure breeds, we performed differential gene expression analyses on the subset of the. - The ovine transcriptome was downloaded from the Ensembl database, and all of the bam files that originally mapped to the ovine reference genome were queried against the transcriptome to count the reads belonging to exonic regions. - Similarly, KEGG pathways representing at least four DEGs and 4% of the given pathway were retrieved. - F1 crosses with the flushing diet) of the 12 comparisons were identified. - Therefore, all of the DEGs were grouped, and the unique Ensembl IDs were selected to determine the number of genes that lacked annotations. - SAMtools [72] was used to call SNPs from sorted bam files of the mRNA-seq data. - After SNP calling, low- quality SNPs (quality less than 10) and SNPs that appeared at more than two times the average depth coverage of the samples were removed. - The average depth coverage for each sample was calculated using bedtools [73], and the mean of the average depth cover- age for all samples was used for filtration. - VEP predicts the consequence of variants on genomic regions and the locations of the variants. - The VCF file consisting of the SNPs for all samples was used for the annotation against the ovine genome. - miRNA variants were analysed using GATK [76], which forms part of the CAP-miRSeq pipeline. - The SNVs located in the seed regions of the mature miRNA were considered for further annotation. - The details of the RNA extraction are provided as supplementary methods (see Additional file 1).. - Real-time PCR primers were designed on the basis of the mRNA sequences of the selected 5 candidate genes available in the GenBank database using Primer3 Express version 4.0.0 software [76] (http://primer3.wi.- mit.edu. - Compositions of the feed in the feeding trial. - Effect of flushing on the body condition scores of the ewes. - Summary of the mRNA-seq data. - Summary of the miRNA-Seq data. - (B) Principal component analysis (PCA) plot of the top 500 differentially expressed genes. - Venn diagram showing the overlap of the top 500 genes among the breeds.. - Additional file 2: Summary of sheep phenotype records that also includes all of the different blood plasma measurements. - The funding bodies do not have any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - Pulsatile release of LH and secretion of ovarian steroids in sheep during the luteal phase of the estrous cycle.. - Review of the effects of the Booroola gene (FecB) on sheep production. - Investigation of prolific sheep from UK and Ireland for evidence on origin of the mutations in BMP15 (FecX(G), FecX(B)) and GDF9 (FecG(H)) in Belclare and Cambridge sheep
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