A sheep pangenome reveals the spectrum of structural variations and their effects on tail phenotypes.

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5' untranslated region (5' UTR) of HOXB13 is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.

Long divergent haplotypes introgressed from wild sheep are associated with distinct morphological and adaptive characteristics in domestic sheep.

The worldwide sheep population comprises more than 1000 breeds. Together, these exhibit a considerable morphological diversity, which has not been extensively investigated at the molecular level. Here, we analyze whole-genome sequencing individuals of 1,098 domestic sheep from 154 breeds, and 69 wild sheep from seven Ovis species. On average, we detected 6.8%, 1.0% and 0.2% introgressed sequence in domestic sheep originating from Iranian mouflon, urial and argali, respectively, with rare introgressions from other wild species. Interestingly, several introgressed haplotypes contributed to the morphological differentiations across sheep breeds, such as a RXFP2 haplotype from Iranian mouflon conferring the spiral horn trait, a MSRB3 haplotype from argali strongly associated with ear morphology, and a VPS13B haplotype probably originating from urial and mouflon possibly associated with facial traits. Our results reveal that introgression events from wild Ovis species contributed to the high rate of morphological differentiation in sheep breeds, but also to individual variation within breeds. We propose that long divergent haplotypes are a ubiquitous source of phenotypic variation that allows adaptation to a variable environment, and that these remain intact in the receiving population probably due to reduced recombination.

TGIF1 and SF1 polymorphisms are associated with litter size in Small Tail Han sheep.

TGF-ß induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3'UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3'UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs.

Identification of Prolificacy-Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics.

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5'-3' exoribonuclease 2 (XRN2) in PF versus MF, and bcl-2-associated athanogene 4 (BAG4), insulin-like growth factor-1 receptor (IGF1R), hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin-releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.

Polymorphisms of the melatonin receptor 1A gene that affects the reproductive seasonality and litter size in Small Tail Han sheep.

Previous researches have shown that MTNR1A plays an essential role in sheep reproduction. However, most researches focused more on the reproductive seasonality of sheep, and few scientists had studied the association of polymorphisms of the MTNR1A gene with ovine litter size and reproductive seasonality. Therefore, we chose MTNR1A gene to detect its novel sequence polymorphisms and population genetics and analyse their association with seasonal reproduction and litter size in ewes. The mRNA expression level in hypothalamus, pituitary and ovary was also detected. In this study, five polymorphisms (g.15118664G > T, g.15118683C > T, g.15118756C > T, g.15118774C > T and g.15118951G > A) were identified in exon 2. Most importantly, the g.15118683C > T and g.15118951G > A were significant difference between year-round oestrous sheep and seasonal oestrous sheep (p < .01), and g.15118756C > T had a great effect on litter size of Small Tail Han sheep (p < .05). In addition, the mRNA expression level of MTNR1A in the hypothalamus of polytocous Small Tail Han sheep was significantly higher than that in monotocous Small Tail Han sheep (p < .01) and the expression of MTNR1A in the hypothalamus of year-round oestrous sheep was significantly higher than that in seasonal oestrous sheep (p < .01). Polymorphisms in exon 2 may regulate the reproductive seasonality and litter size of ewes by influencing gene expression to regulate the reproductive seasonality and litter size of ewes. Our studies provided useful guidance in marker-assisted selection of the litter size in Small Tail Han sheep.

Design and characterization of a high-resolution multiple-SNP capture array by target sequencing for sheep.

The efficiency of molecular breeding largely depends on inexpensive genotyping arrays. In this study, we aimed to develop an ovine high-resolution multiple-single-nucleotide polymorphism (SNP) capture array, based on genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology. All the markers were from 40K captured regions, including genes located within selective sweep regions, breed-specific regions, quantitative trait loci (QTL), and the potential functional SNPs on the sheep genome. The results showed that a total of 210K high-quality SNPs were identified in the 40K regions, indicating a high average capture ratio (99.7%) for the target genomic regions. Using genotyped data (n = 317) from liquid chip technology, we further performed genome-wide association studies (GWAS) to detect the genetic loci affecting sheep hair types and teat number. A single significant association signal for hair types was identified on 6.7-7.1 Mb of chromosome 25. The IRF2BP2 gene (chr25: 7,067,974-7,071,785), which is located within this genomic region, has been previously known to be involved in hair/wool traits in sheep. The results further showed a new candidate region around 26.4 Mb of chromosome 13, between the ARHGAP21 and KIAA1217 genes, that was significantly related to teat number in sheep. The haplotype patterns of this region also showed differences in animals with 2, 3, or 4 teats. Advances in using the high-accuracy and low-cost liquid chip are expected to accelerate sheep genomic and breeding studies in the coming years.

Large-scale genotyping platforms are valuable tools for animal selection and breeding programs. The bead chip has been widely used in both research and commercial applications for a long time. A highly efficient and economical genotyping platform has been developed recently. In the present study, by combining the advantages of resequencing and bead chips, we developed a high-resolution capture array based on target sequencing with capture-in-solution technology (liquid chip), including updated functional probes according to the latest research. We further evaluated this approach by using 317 individuals and found that 210K single-nucleotide polymorphisms can be accurately genotyped, confirming the ratio of the captured regions compared with the designed rations is around 99.7%. Genome-wide association studies conducted using this chip suggested IRF2BP2 gene may be involved in hair types and ARHGAP21-KIAA1217 locus may be related to teats number. The liquid chip with high accuracy and low cost can be widely used in genome-wide association studies and genome selection, supporting efforts in molecular breeding and genetic improvement of sheep.

Expression, structure and function analysis of the sperm-oocyte fusion genes Juno and Izumo1 in sheep (Ovis aries).

BACKGROUND: JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization. METHODS: PCR was used to clone the full-length DNA sequence of the Juno gene in sheep. The single nucleotide polymorphism (SNP) loci of Juno were genotyped by Sequenom MassARRAY®. PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1. Reverse transcriptase-PCR (RT-PCR) and real time-quantitative-PCR (RT-qPCR) were used to analyze the genes' expression in tissues of sheep, and single cell RNA-seq was used to analyze the genes' expression in oocytes, granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes. Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins. RESULTS: The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened. We found a significant association between the g.848253 C > A locus of Juno and litter size of Small Tail Han sheep (P < 0.05). The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained. We found a new segment of the Izumo1 CDS consisting of 35 bp, and we confirmed the Izumo1 gene has 9 exons, not 8. RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues, respectively (P < 0.01). Single cell RNA-seq showed Juno was specifically expressed in oocytes, but not in granulosa cells or follicular theca, while Izumo1 displayed little to no expression in all three cell types. There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes, indicating expression of Juno is not related to litter size traits. Bioinformatic analysis revealed the g.848253 C > A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence. CONCLUSIONS: For the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.

Polymorphism, expression and structure analysis of key genes in the ovarian steroidogenesis pathway in sheep (Ovis aries).

BACKGROUND: Litter size is an important factor that significantly affects the development of the sheep industry. Our previous TMT proteomics analysis found that three key proteins in the ovarian steroidogenesis pathway, STAR, HSD3B1, and CYP11A1, may affect the litter size trait of Small Tail Han sheep. OBJECTIVE: The purpose of this study was to better understand the relationship between polymorphisms of these three genes and litter size. MATERIAL AND METHOD: Sequenom MassARRAY detected genetic variance of the three genes in 768 sheep. Real-time qPCR of the three genes was used to compare their expression in monotocous and polytocous sheep in relevant tissues. Finally, bioinformatics analysis predicted the protein sequences of the different SNP variants. RESULT: Association analysis showed that there was a significant difference in litter size among the genotypes at two loci of the CYP11A1 gene (p < 0.05), but no significant difference was observed in litter size among all genotypes at all loci of the STAR and HSD3B1 genes (p > 0.05). However, STAR expression was significantly different in polytocous and monotocous sheep in the pituitary (p < 0.01). Tissue-specific expression in the ovary was observed for HSD3B1 (p < 0.05), but its expression was not different between polytocous and monotocous sheep. Bioinformatics analysis showed that the g.33217408C > T mutation of CYP11A1 resulted in major changes to the secondary and tertiary structures. In contrast, gene polymorphisms in STAR and HSD3B1 had minimal impacts on their protein structures. DISCUSSION: This may explain why the CYP11A1 variant impacted litter size while the others did not. The single nucleotide polymorphism of the CYP11A1 gene would serve as a good molecular marker when breeding to increase litter size in sheep. Our study provides a basis for further revealing the function of the ovarian steroidogenesis pathway in sheep reproduction and sheep breeding.

The effect of SNP rs400827589 in exon 2 of the MTNR1B gene on reproductive seasonality and litter size in sheep.

In mammals, the melatonin receptor gene has been widely studied since it has a great influence on reproductive traits. However, little is known about the association between polymorphism of the coding region of the MTNR1B gene and year-round oestrus or the litter size in Small Tail Han sheep. To better understand the effects of single nucleotide polymorphism (SNP) rs400827589 in MTNR1B, a population polymorphism analysis was conducted using genotyping data in 45 sheep breeds around the world. The results indicated that TT was the dominant genotype in all sheep breeds. The associations of this SNP with reproductive seasonality and litter size in Small Tail Han sheep showed rs400827589 was correlated with fecundity as assessed by reproductive seasonality and litter size (p < .05). Bioinformatics analysis indicated the change in amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. The present results demonstrate that rs400827589 could be used in the marker-assisted selection of the litter size in Small Tail Han sheep.

Mutations in NLRP5 and NLRP9 are Associated with Litter Size in Small Tail Han Sheep.

Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY® SNP system at the loci g.60495375A > G, g.60495363G > A, and g.60499690C > A in NLRP5, and g.59030623T > C and g.59043397A > C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy-Weinberg equilibrium (p > 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G > A in NLRP5 and g.59030623T > C in NLRP9, were highly associated with litter size. The mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T > C may enhance the combining capacity of NLRP9 with these proteins; consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals.

Identification and Characterization of Hypothalamic Alternative Splicing Events and Variants in Ovine Fecundity-Related Genes.

Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small-tailed Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as LGALS3 in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.

Polymorphisms in the ASMT and ADAMTS1 gene may increase litter size in goats.

Prolificacy of most local goat breeds in China is low. Jining Grey goat is one of the most prolific goat breeds in China, it is an important goat breed for the rural economy. ASMT (acetylserotonin O-methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are essential for animal reproduction. Single nucleotide polymorphisms (SNPs) of ASMT and ADAMTS1 genes in the highly prolific breed (Jining Grey goats), medium prolific breed (Boer goats and Guizhou White goats) and low prolific breeds (Angora goats, Liaoning Cashmere goats and Inner Mongolia Cashmere goats) were detected by polymerase chain reaction-restriction fragment length polymorphism and sequencing. Two SNPs (g.158122T>C, g.158700G>A) of ASMT gene and two SNPs (g.7979798A>G, g.7979477C>T) of ADAMTS1 gene were identified. For g.158122T>C of ASMT gene, further analysis revealed that genotype TC or CC had 0.66 (p < 0.05) or 0.75 (p < 0.05) kids more than those with genotype TT in Jining Grey goats. No significant difference (p > 0.05) was found in litter size between TC and CC genotypes. The SNP (g.158122T>C) caused a p.Tyr298His change and this SNP mutation resulted in changes in protein binding sites and macromolecule-binding sites. The improvement in reproductive performance may be due to changes in the structure of ASMT protein. For g.7979477C>T of ADAMTS1 gene, Jining Grey does with genotype CT or TT had 0.82 (p < 0.05) or 0.86 (p < 0.05) more kids than those with genotype CC. No significant difference (p > 0.05) was found in litter size between CT or TT genotypes. These results preliminarily indicated that C allele (g.158122T>C) of ASMT gene and T allele (g.7979477C>T) of ADAMTS1 gene are potential molecular markers which could improve litter size of Jining Grey goats and be used in goat breeding.

Comparative Transcriptomics Identify Key Hypothalamic Circular RNAs that Participate in Sheep (Ovis aries) Reproduction.

Circular RNA (circRNA), as an emerging class of noncoding RNA, has been found to play key roles in many biological processes. However, its expression profile in the hypothalamus, a powerful organ initiating the reproductive process, has not yet been explored. Therefore, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the FecB ++ genotype. We totally identified 41,863 circRNAs from sheep hypothalamus, in which 333 (162 were upregulated, while 171 were downregulated) were differentially expressed in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), moreover, 340 circRNAs (163 were upregulated, while 177 were downregulated) were differentially expressed in polytocous sheep in the luteal phase versus monotocous sheep in the luteal sheep (PL vs. ML). We also identified several key circRNAs including oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, oar_circ_0010234, and oar_circ_0013788 through functional enrichment analysis and oar_circ_0012110 through a competing endogenous RNA network, most of which may participate in reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression, indirectly or directly. Our study explored the overall expression profile of circRNAs in sheep hypothalamus, which potentially provides an alternative insight into the mechanism of sheep prolificacy without the effects of FecB mutation.

Single nucleotide polymorphisms in BMP2 and BMP7 and the association with litter size in Small Tail Han sheep.

Although it has been investigated for many years, the physiological processes regulating prolificacy in sheep remains unclear because of regulation by many genes. To better understand the effects of three single nucleotide polymorphisms (SNPs) comprising g.48462350C>T in BMP2, g.58171856C>G and g.58171886A>C in BMP7, a population genetic analysis was conducted using data obtained from genotyping in 768 sheep from six breeds (three polytocous and three monotocous). The results indicate that all the sheep breeds were considered to conform to the Hardy-Weinberg equilibrium (P > 0.05). The associations of these three SNPs with litter size in 384 Small Tail Han sheep were analyzed, therefore, and found to be correlated with fecundity as assessed by mean litter size (P < 0.05). Bioinformatic analysis indicated there was a transmembrane domain change that occurred after a mutation in BMP2 at g.48462350C>T, and changes involving transcription factors such as USF1, USF2 and INMS1 in the BMP7 promoter region might be involved in greater sheep prolificacy.

Comparative Transcriptomics Reveal Key Sheep (Ovis aries) Hypothalamus LncRNAs that Affect Reproduction.

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.

Integrated Hypothalamic Transcriptome Profiling Reveals the Reproductive Roles of mRNAs and miRNAs in Sheep.

Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., POMC, GNRH1, PRL, GH, TRH, and TTR) and mRNA-miRNAs pairs (e.g., TRH co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and PRL regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropin-releasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release via direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy.