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Background: Sheep horns play a critical role in the survival and reproduction of sheep. Research on sheep horns not only aids in comprehending their biological roles but is also vital for developing hornless breeds. Although previous studies have suggested that KLK7 may be associated with keratin growth, there are few studies that have focused on the role of KLK7 in sheep horns. This study aims to elucidate the relationship between KLK7 and sheep horns by analyzing the expression, genetic polymorphisms, and potential functions of KLK7 in sheep horns. Results: This study utilized RNA sequencing (RNA-Seq) data to analyze the expression levels of the KLK7 gene across different species, as well as among different breeds, tissue types, and genders in sheep. Potential functional sites of KLK7 were explored using whole-genome sequencing (WGS) data. Allele specific expression sites in the KLK7 gene sequence were identified. Finally, the WGS data were linked with sheep horn length for association analysis, and significantly different single-nucleotide polymorphisms (SNPs) were detected and validated by Kompetitive Allele Specific PCR (KASP) genotyping. Conclusion: Our results demonstrate that KLK7 was highly expressed in soft horn and skin tissues, and its expression was significantly higher in small-horned sheep than it was in large-horned sheep, suggesting that KLK7 may have an inhibitory effect on horn growth. By comparing the amino acid sequence of KLK7 with KLK7 sequences in other species, we discovered eight amino acids at specific positions in the KLK7 protein sequence that may have regulatory functions in determining the size of horns in ruminant animals. Thirteen SNPs with F-statistic value (Fst) > 0.15 were identified. By integrating RNA-Seq and WGS data, we discovered two SNPs (g.56695395 T > C and g.56695484A > C) with allele specific expression between the large- and small-horned sheep. The two SNPs were validated and were found to have significantly different (P < 0.05) effects on horn length. Our findings suggest a strong association between KLK7 and sheep horn length, indicating the potential role of KLK7 in inhibiting horn growth and providing novel insights into the functionality of KLK7. [ABSTRACT FROM AUTHOR]

The growth and development of horns are primarily controlled by the skin. The ALOX12B gene is crucial for epidermal barrier function and may have a significant impact on horn growth. The purpose of this study was to investigate the expression of ALOX12B across different sheep breeds and tissues by utilizing RNA sequencing. Additionally, potential functional sites were identified in conjunction with whole genome sequencing. Our findings revealed that ALOX12B was highly expressed in the scurred horn group as opposed to the normal horn group (SHE). ALOX12B expression was also notably high in the skin across several species. Eight loci that may influence horn size were indicated in this study. Through the alignment of the ALOX12B protein sequence from 16 species, 15 amino acid sites were identified specifically expressed in horned animals. In conclusion, this study established a connection between ALOX12B and horn size and identified a series of functional sites that may serve as molecular markers for reducing the presence of horns in Chinese sheep breeds. [ABSTRACT FROM AUTHOR]

The genetic basis of complex traits and phenotypic differentiation remains unclear in pigs. Using nine genomes—seven of which were newly generated, high‐quality de novo assembled genomes—and 1081 resequencing genomes, we built a pan‐genome and identified 134.24 Mb nonredundant nonreference sequences, 1099 novel protein‐coding genes, 187,927 structural variations (SVs) and 30,143,962 single‐nucleotide polymorphisms (SNPs). Analysis of selective domestication revealed BRCA1 associated with enhanced adipocyte growth and fat deposition, and ABCA3 linked to an alleviated immune response and reduced lung injury. Integrating 162 transcriptomes and 162 methylomes of skeletal muscle across 27 developmental stages revealed the regulatory mechanism of phenotypic differentiation between Eastern and Western breeds. Artificial selection reshaped local DNA methylation status and imparted regulatory effects on the progression patterns of heterochronic genes such as GHSR and BDH1, particularly during embryonic development. Altogether, our work provides valuable resources for understanding molecular mechanisms behind phenotypic variations and enhancing the genetic improvement programs in pigs. Highlights: Combining nine high‐quality genomes and 1081 whole‐genome resequencing data, a relatively complete pan‐genome and genetic variation data set of pigs was constructed, laying the foundation for analyzing the complex traits of pigs.Abundant signals and genes of selective sweeps related to pig fat deposition, muscle growth, and immune traits were discovered, providing key genetic markers for future breeding programs.Integration of transcriptome and methylome data across 27 skeletal muscle development stages revealed the epigenetic and heterochronic gene regulatory mechanisms of skeletal muscle growth differences between Eastern and Western pig breeds.Artificial selection has a significant impact on DNA methylation and heterochronic gene regulation, which is closely related to the differences in skeletal muscle growth and meat quality between Eastern and Western pig breeds. [ABSTRACT FROM AUTHOR]

The quality of meat is important to the consumer. Color is a primary indicator of meat quality and is characterized mainly into lightness, redness, and yellowness. Here, we used the genome-wide association study (GWAS) and gene-based association analysis with whole-genome resequencing of 230 fast-growing white-feathered chickens to map genes related to meat lightness and redness to a 6.24 kb QTL region (Chr15: 6298.34–6304.58 kb). This analysis revealed that only the protein phosphatase 1 catalytic subunit gamma (PPP1CC) was associated with meat color (P = 8.65E-08). The causal relationships between PPP1CC expression and meat lightness/redness were further validated through Mendelian randomization analyses (P < 2.9E-12). Inducible skeletal muscle-specific PPP1CC knockout (PPP1CC-SSKO) mice were generated and these mice showed increased lightness and decreased myoglobin content in the limb muscles. In addition, the predominant myofiber shifted from slow-twitch to fast-twitch myofibers. Through transcriptome and targeted metabolome evidence, we found that inhibition of PPP1CC decreased the expression of typical slow-twitch myofiber and myofiber-type specification genes and enhanced the glycolysis pathway. Functional validation through a plasmid reporter assay revealed that a SNP (rs315520807, C > T) located in the intron of PPP1CC could regulate the gene transcription activity. The differences in meat color phenotypes, myoglobin content, frequency of rs315520807 variant, expression of PPP1CC and fast-twitch fiber marker genes were detected between fast-growing white-feathered chickens and local chickens. In this study, PPP1CC was identified as the causative gene for meat color, and the novel target gene and variant that can aid in the innovation of meat improvement technology were detected. Author summary: Color is a primary indicator of the biochemical and physiological properties of meat and affects consumer perception. Meat color is characterized mainly into lightness, redness and yellowness. The causative gene of meat lightness and redness has not yet been identified. We found that the expression of protein phosphatase 1 catalytic subunit gamma (PPP1CC) was causally related to breast muscle lightness and redness in fast-growing white-feathered chickens using a GWAS, gene-based association analysis and Mendelian randomization analysis. Using mice with inducible, skeletal muscle-specific knockout of PPP1CC (PPP1CC-SSKO), we also revealed that lightness increased and myoglobin content decreased in the limb muscle and identified that myofiber-type specification is one of the mechanisms by which PPP1CC influences meat color. We subsequently discovered an intron variant (rs315520807, C > T) in PPP1CC that could regulate the gene transcription. The causative gene and potential causative variant for chicken lightness and redness could be utilized in the innovation of meat improvement technology. [ABSTRACT FROM AUTHOR]

Under the current context of intensive farming, small-horned animals are more suitable for large-scale breeding. The CASP14 gene, closely associated with skin and keratin formation, may influence horn size due to its link with skin development. This study comprehensively analyzed the tissue-specific expression of CASP14 using RNA-Seq data, identified functional sites through whole-genome sequencing (WGS), and investigated allele-specific expression (ASE) validated by KASP assays. Results showed significantly higher CASP14 expression in the scurred group com-pared to the SHE group, with pronounced expression in the skin. Interbreed comparisons also revealed elevated CASP14 levels in the scurred group. Analysis of potential functional sites indicated structural similarities in the CASP14 protein among horned mammals. Five ASE events were discovered, and intersecting these with SNPs and high fixation index loci (Fst > 0.05) identified three potential functional sites: 7941628, 7941817, and 7941830. The SNP site 7944295 was selected for T-test analysis and further validated through KASP assays, corroborating the role of CASP14 in sheep horn phenotypes. Our findings suggest that CASP14 plays a significant role in horn development, offering insights into breeding strategies for small-horned animals. [ABSTRACT FROM AUTHOR]

Milk production is an important trait that influences the economic value of camels. However, the genetic regulatory mechanisms underlying milk production in camels have not yet been elucidated. We aimed to identify candidate molecular markers that affect camel milk production. We classified Junggar Bactrian camels (9–10-year-old) as low-yield (<1.96 kg/d) or high-yield (>2.75 kg/d) based on milk production performance. Milk fat (5.16 ± 0.51 g/100 g) and milk protein (3.59 ± 0.22 g/100 g) concentrations were significantly lower in high-yielding camels than those in low-yielding camels (6.21 ± 0.59 g/100 g, and 3.93 ± 0.27 g/100 g, respectively) (p < 0.01). There were no apparent differences in gland tissue morphology between the low- and high-production groups. Whole-genome resequencing of 12 low- and 12 high-yield camels was performed. The results of selection mapping methods, performed using two methods (FST and θπ), showed that 264 single nucleotide polymorphism sites (SNPs) overlapped between the two methods, identifying 181 genes. These genes were mainly associated with the regulation of oxytocin, estrogen, ErbB, Wnt, mTOR, PI3K-Akt, growth hormone synthesis/secretion/action, and MAPK signaling pathways. A total of 123 SNPs were selected, based on significantly associated genomic regions and important pathways for SNP genotyping, for verification in 521 additional Bactrian camels. This analysis showed that 13 SNPs were significantly associated with camel milk production yield and 18 SNPs were significantly associated with camel milk composition percentages. Most of these SNPs were located in coding regions of the genome. However, five and two important mutation sites were found in the introns of CSN2 (β-casein) and CSN3 (κ-casein), respectively. Among the candidate genes, NR4A1, ADCY8, PPARG, CSN2, and CSN3 have previously been well studied in dairy livestock. These observations provide a basis for understanding the molecular mechanisms underlying milk production in camels as well as genetic markers for breeding programs aimed at improving milk production. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigated the relationship between the BMPR1A gene and horn type in sheep. The study found that the expression of the BMPR1A gene was significantly higher in the normal horn sheep compared to scurred sheep. This trend was observed in multiple sheep breeds. It was also discovered that there is high expression in the skin of three species: cattle, sheep, and pigs. Importantly, it was found that certain nucleotides in the BMPR1A gene are likely to be crucial in horn size and horn type. Sheep horns are composed of bone and sheaths, and the BMPR1A gene is required for cartilage and osteogenic differentiation. Therefore, the BMPR1A gene may have a function related to the sheep horn, but its relationship with the sheep horn remains unclear. In this study, we first utilized RNA sequencing (RNA-seq) data to investigate the expression of the BMPR1A gene in different tissues and breeds of sheep. Second, whole-genome sequencing (WGS) data were used to explore the functional sites of the BMPR1A gene. Lastly, the allele-specific expression of the BMPR1A gene was explored. Our results indicate that BMPR1A gene expression is significantly higher in the normal horn groups than in the scurred groups. Importantly, this trend is consistent across several sheep breeds. Therefore, this finding suggests that the BMPR1A gene may be related to horn type. A total of 43 Single-Nucleotide Polymorphisms (SNPs) (F-statistics > 0.15) and 10 allele-specific expressions (ASEs) exhibited difference between the large and small horn populations. It is probable that these sites significantly impact the size of sheep horns. Compared to other polled species, we discovered ten amino acid sites that could influence horn presence. By combining RNA-seq and WGS functional loci results, we identified a functional site at position 40574836 on chromosome 25 that is both an SNP and exhibits allele-specific expression. In conclusion, we demonstrated that the BMPR1A gene is associated with horn type and identified some important functional sites which can be used as molecular markers in the breeding of sheep horns. [ABSTRACT FROM AUTHOR]

Blood samples are easily obtained from sheep. Therefore, blood analysis can be a convenient method for evaluating reproductive traits in sheep by detecting genetic and metabolic changes in the ovary. By combining 167 RNA sequencing data and 60 untargeted metabolomics data, this study analyzed the relationship between genes and metabolites in the ovary and blood. The conjoint KEGG enrichment analysis enriched glutathione (GSH) metabolic pathways both in the ovary and blood. This finding provides an explanation for possible GSH metabolic processes in the ovary with metabolite exchange in the blood. The metabolite–gene–disease interaction network revealed a correlation between the expression of certain Bardet–Biedl syndrome (BBS) family genes in the ovary and blood. This indicates that BBS family genes, such as BBS10 in sheep blood, could be a potential biomarker for BBS. We investigated the relationship between BBS10 gene expression in the ovary and lambing numbers using whole-genome sequencing data from 450 ewes. Our findings suggest that g.112314188C>G may lead to decreased litter size in ewes carrying the FecB gene. These single nucleotide polymorphisms could be potential molecular markers for breeding sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: FBN1, ZFAT and FAM184B have been screened as candidate genes for the reproduction of sheep. Therefore, it is necessary to verify these genes in the population and determine the associated loci for litter size. The association of litter size with the genotypes of three candidate genes was analyzed using the fixed effects model in Xinggao mutton sheep. The results showed that the g.160338382 T > C in FBN1 was significantly associated with litter size in Xinggao mutton sheep and that this effect was independent of the FecB mutation. Overall, this study provides a useful genetic marker for improving sheep fecundity. Previous studies have screened key candidate genes for litter size in sheep, including fibrillin-1 (FBN1), family with sequence similarity 184 member B (FAM184B) and zinc finger and AT-hook domain containing (ZFAT). Therefore, it is necessary to verify these genes in the Xinggao mutton sheep population and determine the associated loci for litter size. In this study, three loci (FBN1 g.160338382 T > C, FAM184B g.398531673 C > T and ZFAT g.20150315 C > T) were firstly screened based on the population differentiation coefficient between the polytocous and monotocous sheep groups. Then, population genetic analysis and association analysis were performed on these loci. The results revealed that the g.160338382 T > C in FBN1 was significantly associated with the litter size of sheep. Moreover, there was no significant interaction effect between the g.160338382 T > C locus and FecB on litter size. Notably, g.160338382 T > C is adjacent to the anterior border of exon 58 and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and ultimately cause changes in the structure and function of the protein. In summary, our results provided a potentially effective genetic marker for improving the litter size of sheep. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs (n = 3,087,929) in pigs and found that young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs. A catalog of over 3 million transposable elements in pigs reveals that evolutionarily young SINEs are associated with increased epigenomic silencing, and may be related to expression of nearby genes. [ABSTRACT FROM AUTHOR]

Background: Beef tenderness is a complex trait of economic importance for the beef industry. Understanding the epigenetic mechanisms underlying this trait may help improve the accuracy of breeding programs. However, little is known about epigenetic effects on Bos taurus muscle and their implications in tenderness, and no studies have been conducted in Bos indicus. Results: Comparing methylation profile of Bos indicus skeletal muscle with contrasting beef tenderness at 14 days after slaughter, we identified differentially methylated cytosines and regions associated with this trait. Interestingly, muscle that became tender beef had higher levels of hypermethylation compared to the tough group. Enrichment analysis of predicted target genes suggested that differences in methylation between tender and tough beef may affect signal transduction pathways, among which G protein signaling was a key pathway. In addition, different methylation levels were found associated with expression levels of GNAS, PDE4B, EPCAM and EBF3 genes. The differentially methylated elements correlated with EBF3 and GNAS genes overlapped CpG islands and regulatory elements. GNAS, a complex imprinted gene, has a key role on G protein signaling pathways. Moreover, both G protein signaling pathway and the EBF3 gene regulate muscle homeostasis, relaxation, and muscle cell-specificity. Conclusions: We present differentially methylated loci that may be of interest to decipher the epigenetic mechanisms affecting tenderness. Supported by the previous knowledge about regulatory elements and gene function, the methylation data suggests EBF3 and GNAS as potential candidate genes and G protein signaling as potential candidate pathway associated with beef tenderness via methylation. [ABSTRACT FROM AUTHOR]

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating 47 human genome-wide association studies, we demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases. Annotating functional elements of the genome helps the interpretation of genetic variation. Here, the authors compile functional genomics data for the pig genome over 14 tissues with 15 different chromatin states, integrate the data with WGS and GWAS data, and compare conservation of regulatory elements across mouse and human tissues. [ABSTRACT FROM AUTHOR]

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources. In order to interpret non-coding variants, information about regulatory elements in the genome is essential. Here, the authors annotate regulatory elements in chicken, pig and cattle, and characterize conservation of these elements between species. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the genetic diversity and phylogenetic relationship of nine sheep populations, including two famous high prolific populations and seven popular mutton populations raised in China. Overall, these sheep populations in this study exhibited a rich genetic diversity. Both the expected heterozygosity and Nei's unbiased gene diversity ranged from 0.64 to 0.75, with the lowest value found in Dorset sheep (DST) and the highest in Hu sheep (HUS) and Ba Han sheep (BAS). The polymorphic information content (PIC) varied between 0.59 in DST and 0.71 in HUS and BAS. Specifically, for individual breeds, the small-tail Han sheep (STH) and the four introduced populations did not display the expected diversity; therefore more attention should be paid to the maintenance of diversity during management of these populations. The results of un-weighted pair-group method (UPGMA) phylogenetic tree and structure analysis indicated that the nine investigated populations can be divided into two groups. Suffolk (SUF) and DST were clustered in one group, and the other group can be further divided into three clusters: German Mutton Merino (GMM)–BAS–Bamei Mutton sheep (BAM), HUS–STH and Du Han (DOS)–Dorper (DOP). This clustering result is consistent with sheep breeding history. TreeMix analysis also hinted at the possible gene flow from GMM to SUF. Together, an in-depth view of genetic diversity and genetic relationship will have important implications for breed-specific management. [ABSTRACT FROM AUTHOR]

Simple Summary: Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. There is large variation in the reproductive abilities of Bamei mutton sheep. After recent artificial selection, the average lambing rate of the Bamei mutton nucleus group was over 150%. We used the FST (Fixation Index) and XP-EHH (The Cross-Population Extended Haplotype Homozygosity) statistical approach to detect the selective sweeps between high- and low-fecundity Bamei mutton sheep groups. JUN (JUN proto-oncogene, AP-1 transcription factor subunit), ITPR3 (inositol 1,4,5-trisphosphate receptor type 3, PLCB2 (phospholipase C beta 2), HERC5 (HECT and RLD domain containing E3 ubiquitin protein ligase 5), and KDM4B (lysine demethylase 4B) were detected that are potential responsible for litter size. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection. [ABSTRACT FROM AUTHOR]

Background: Sheep have developed the ability to store fat in their tails, which is a unique way of reserving energy to survive a harsh environment. However, the mechanism underlying this adaptive trait remains largely unsolved. Results: In the present study, we provide evidence for the genetic determinants of fat tails, based on whole genome sequences of 89 individual sheep. A genome-wide scan of selective sweep identified several candidate loci including a region at chromosome 13, a haplotype of which underwent rapid evolution and spread through fat-tailed populations in China and the Middle East. Sequence analysis revealed an inter-genic origin of this locus, which later became a hotspot of ruminant-specific retro-transposon named BovB. Additionally, the candidate locus was validated based on a fat- and thin-tailed cross population. The expression of an upstream gene BMP2 was differentially regulated between fat-tailed and thin-tailed individuals in tail adipose and several other tissue types. Conclusions: Our findings suggest the fixation of fat tails in domestic sheep is caused by a selective sweep near a retro-transposable hotspot at chromosome 13, the diversity of which specifically affects the expression of BMP2. The present study has shed light onto the understanding of fat metabolism. [ABSTRACT FROM AUTHOR]

Simple Summary: Growth differentiation factor 9 (GDF9) is an important gene for ovine fertility. GDF9 is highly expressed in the ovary as opposed to other tissues, but the reason for this is unknown. Our study found this can be caused by the methylation level of the promoter CpG island mC-4 site. This finding contributes to the understanding of the regulatory mechanism of GDF9 gene in reproduction. Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression. [ABSTRACT FROM AUTHOR]

Simple Summary: Litter size is one of the most important reproductive traits in sheep. Two single nucleotide polymorphisms (SNPs), g.71874104G>A and g.71833755T>C, in the Histone Cell Cycle Regulator (HIRA) gene, were identified by whole-genome sequencing (WGS) and may be correlated with litter size in sheep. The two SNPs were genotyped and expression patterns of HIRA was determined in sheep breeds with different fecundity and in groups of Small Tail Han sheep producing large or small litters. Association analysis indicated that both SNPs were significantly correlated with litter size in Small Tail Han sheep. Furthermore, high levels of HIRA expression may have a negative effect on litter size in Small Tail Han sheep. Maintenance of appropriate levels of fecundity is critical for efficient sheep production. Opportunities to increase sheep litter size include identifying single gene mutations with major effects on ovulation rate and litter size. Whole-genome sequencing (WGS) data of 89 Chinese domestic sheep from nine different geographical locations and ten Australian sheep were analyzed to detect new polymorphisms affecting litter size. Comparative genomic analysis of sheep with contrasting litter size detected a novel set of candidate genes. Two SNPs, g.71874104G>A and g.71833755T>C, were genotyped in 760 Small Tail Han sheep and analyzed for association with litter size. The two SNPs were significantly associated with litter size, being in strong linkage disequilibrium in the region 71.80–71.87 Mb. This haplotype block contains one gene that may affect litter size, Histone Cell Cycle Regulator (HIRA). HIRA mRNA levels in sheep with different lambing ability were significantly higher in ovaries of Small Tail Han sheep (high fecundity) than in Sunite sheep (low fecundity). Moreover, the expression levels of HIRA in eight tissues of uniparous Small Tail Han sheep were significantly higher than in multiparous Small Tail Han sheep (p < 0.05). HIRA SNPs significantly affect litter size in sheep and are useful as genetic markers for litter size. [ABSTRACT FROM AUTHOR]