Sheep Genome Research Articles

Ancient hybridization patterns between bighorn and thinhorn sheep.

Whole-genome sequencing has advanced the study of species evolution, including the detection of genealogical discordant events such as ancient hybridization and incomplete lineage sorting (ILS). The evolutionary history of bighorn (Ovis canadensis) and thinhorn (Ovis dalli) sheep present an ideal system to investigate evolutionary discordance due to their recent and rapid radiation and putative secondary contact between bighorn and thinhorn sheep subspecies, specifically the dark pelage Stone sheep (O.dalli stonei) and predominately white Dall sheep (O.dalli dalli), during the last ice age. Here, we used multiple genomes of bighorn and thinhorn sheep, together with snow (O.nivicola) and the domestic sheep (O.aries) as outgroups, to assess their phylogenomic history, potential introgression patterns and their adaptive consequences. Among the Pachyceriforms (snow, bighorn and thinhorn sheep) a consistent monophyletic species tree was retrieved; however, many genealogical discordance patterns were observed. Alternative phylogenies frequently placed Stone and bighorn as sister clades. This relationship occurred more often and was less divergent than that between Dall and bighorn. We also observed many blocks containing introgression signal between Stone and bighorn genomes in which coat colour genes were present. Introgression signals observed between Dall and bighorn were more random and less frequent, and therefore probably due to ILS or intermediary secondary contact. These results strongly suggest that Stone sheep originated from a complex series of events, characterized by multiple, ancient periods of secondary contact with bighorn sheep.

Whole-genome sequence analysis unveils different origins of European and Asiatic mouflon and domestication-related genes in sheep

The domestication and subsequent development of sheep are crucial events in the history of human civilization and the agricultural revolution. However, the impact of interspecific introgression on the genomic regions under domestication and subsequent selection remains unclear. Here, we analyze the whole genomes of domestic sheep and their wild relative species. We found introgression from wild sheep such as the snow sheep and its American relatives (bighorn and thinhorn sheep) into urial, Asiatic and European mouflons. We observed independent events of adaptive introgression from wild sheep into the Asiatic and European mouflons, as well as shared introgressed regions from both snow sheep and argali into Asiatic mouflon before or during the domestication process. We revealed European mouflons might arise through hybridization events between a now extinct sheep in Europe and feral domesticated sheep around 6000–5000 years BP. We also unveiled later introgressions from wild sheep to their sympatric domestic sheep after domestication. Several of the introgression events contain loci with candidate domestication genes (e.g., PAPPA2, NR6A1, SH3GL3, RFX3 and CAMK4), associated with morphological, immune, reproduction or production traits (wool/meat/milk). We also detected introgression events that introduced genes related to nervous response (NEURL1), neurogenesis (PRUNE2), hearing ability (USH2A), and placental viability (PAG11 and PAG3) into domestic sheep and their ancestral wild species from other wild species.

42 An Improved, High-quality Ovine Reference Genome Assembly

Abstract Sheep are an important agricultural species used for both food and fiber in the United States and globally. A high-quality reference genome enhances the ability to discover genetic and biological mechanisms influencing important traits, such as meat and wool quality. The rapid advances in genome assembly algorithms and emergence of increasingly long sequence read length provide the opportunity for an improved de novo assembly of the sheep reference genome. Tissue was collected postmortem from an adult Rambouillet ewe selected by USDA-ARS for the Ovine Functional Annotation of Animal Genomes project. Short-read (55x coverage), long-read PacBio (75x coverage), and Hi-C data from this ewe were retrieved from public databases. We generated an additional 50x coverage of Oxford Nanopore data and assembled the combined long-read data with canu v1.9. The assembled contigs were polished with Nanopolish v0.12.5 and scaffolded using Hi-C data with Salsa v2.2. Gaps were filled with PBsuite v15.8.24 and polished with Nanopolish v0.12.5 followed by removal of duplicate contigs with PurgeDups v1.0.1. Chromosomes were oriented by identifying centromeres and telomeres with RepeatMasker v4.1.1, indicating a need to reverse the orientation of chromosome 11 relative to Oar_rambouillet_v1.0. Final polishing was performed with two rounds of a pipeline which consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly has improved continuity (contig N50 of 43.19 Mb) with a 19-fold and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. This significantly improved reference assembly, public at NCBI GenBank under accession number GCA_016772045, will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits relevant the sheep industry.

Whole genome sequencing reveals a complex introgression history and the basis of adaptation to subarctic climate in wild sheep.

To predict species responses to anthropogenic disturbances and climate change, it is reasonable to use species with high sensitivity to such factors. Snow sheep (Ovis nivicola) could represent a good candidate for this; as the only large herbivore species adapted to the cold and alpine habitats of northeastern Siberia, it plays a crucial role in its ecosystem. Despite having an extensive geographical distribution among all ovine species, it is one of the least studied. In this study, we sequenced and analysed six genomes of snow sheep in combination with all other wild sheep species to infer key aspects of their evolutionary history and unveil the genetic basis of their adaptation to subarctic environments. Despite their large census population size, snow sheep genomes showed remarkably low heterozygosity, which could reflect the effect of isolation and historical bottlenecks that we inferred using the pairwise sequential Markovian coalescent and runs of homozygosity. F4 -statistics indicated instances of introgression involving snow sheep with argali (Ovis ammon) and Dall (Ovis dalli) sheep, suggesting that these species might have been more widespread during the Pleistocene. Furthermore, the introgressed segments, which were identified using mainly minimum relative node depth, covered genes associated with immunity, adipogenesis and morphology-related traits, representing potential targets of adaptive introgression. Genes related to mitochondrial functions and thermogenesis associated with adipose tissue were identified to be under selection. Overall, our data suggest introgression as a mechanism facilitating adaptation in wild sheep species and provide insights into the genetic mechanisms underlying cold adaptation in snow sheep.

Identification and expression analysis of the β-defensin genes in the goat small intestine

Defensins represent a family of cysteine-rich peptides that have broad-spectrum antimicrobial activities and serve as a typical kind of effector molecule in the immunity. Ruminant species have a large number of β-defensins in the absence of α- and θ-defensins. It is well-known that the genomes of sheep and cattle harbor at least 43 and 57 β-defensin genes, respectively. However, the repertoire of the goat β-defensin gene family has not been fully elucidated. In this study, we identified a total of 50 β-defensins from the goat genome, including 48 functional genes and 2 pseudogenes. Cross-species genomic and evolutionary analyses showed that all of the β-defensins in goat chromosomes 8, 13 and 23 present one-to-one orthologous relationships to their sheep and cattle counterparts, whereas some β-defensin genes in goat chromosome 27 are goat-specific. Moreover, we observed that some duplicated genes in goat chromosome 27 may be derived from gene copy number variation, and the annotation of sheep and cattle β-defensins appears to be incomplete in the genome. Importantly, real-time PCR analysis showed that 17 β-defensins are expressed in the small intestine with abundant cBD1s expression. These findings significant increased our knowledge of ruminant β-defensin and provided useful information for genetic studies, as well as providing a foundation for future research exploring the role of defensins in the immune response.

Detection and Visualization of Heterozygosity-Rich Regions and Runs of Homozygosity in Worldwide Sheep Populations.

Simple SummaryHeterozygosity-rich regions (HRRs) are regions of high heterozygosity, which can harbor important genes associated with key functional traits such as immune response and disease resilience. Runs of homozygosity (ROH) are contiguous homozygous segments of the genome, which can be informative of the population’s history, structure, demography events, and overall genetic diversity. We first detected factors impacting the identification of ROH and HRR in worldwide sheep populations, which were artificially selected for specific purposes or under natural conditions. We also identified common regions of high homozygosity or heterozygosity among these populations, where a diversity of candidate genes with distinct functions might indicate differential selection pressure on these regions in breeds with different trait expression. Moreover, we evaluated a tool commonly used in the corporate environment, making use of the business intelligence (BI) concept to support managers in the decision-making process, which allowed us to combine results from multiple analyses and create visualization schemes integrating different information. Our findings and proposed tools contribute to the development of more efficient breeding strategies and conservation of genetic resources in sheep and other livestock species.In this study, we chose 17 worldwide sheep populations of eight breeds, which were intensively selected for different purposes (meat, milk, or wool), or locally-adapted breeds, in order to identify and characterize factors impacting the detection of runs of homozygosity (ROH) and heterozygosity-rich regions (HRRs) in sheep. We also applied a business intelligence (BI) tool to integrate and visualize outputs from complementary analyses. We observed a prevalence of short ROH, and a clear distinction between the ROH profiles across populations. The visualizations showed a fragmentation of medium and long ROH segments. Furthermore, we tested different scenarios for the detection of HRR and evaluated the impact of the detection parameters used. Our findings suggest that HRRs are small and frequent in the sheep genome; however, further studies with higher density SNP chips and different detection methods are suggested for future research. We also defined ROH and HRR islands and identified common regions across the populations, where genes related to a variety of traits were reported, such as body size, muscle development, and brain functions. These results indicate that such regions are associated with many traits, and thus were under selective pressure in sheep breeds raised for different purposes. Interestingly, many candidate genes detected within the HRR islands were associated with brain integrity. We also observed a strong association of high linkage disequilibrium pattern with ROH compared with HRR, despite the fact that many regions in linkage disequilibrium were not located in ROH regions.

Comprehensive transcriptome and methylome analysis delineates the biological basis of hair follicle development and wool-related traits in Merino sheep

BackgroundCharacterization of the molecular mechanisms underlying hair follicle development is of paramount importance in the genetic improvement of wool-related traits in sheep and skin-related traits in humans. The Merino is the most important breed of fine-wooled sheep in the world. In this study, we systematically investigated the complexity of sheep hair follicle development by integrating transcriptome and methylome datasets from Merino sheep skin.ResultsWe analysed 72 sequence datasets, including DNA methylome and the whole transcriptome of four gene types, i.e. protein-coding genes (PCGs), lncRNAs, circRNAs, and miRNAs, across four embryonic days (E65, E85, E105, and E135) and two postnatal days (P7 and P30) from the skin tissue of 18 Merino sheep. We revealed distinct expression profiles of these four gene types across six hair follicle developmental stages, and demonstrated their complex interactions with DNA methylation. PCGs with stage-specific expression or regulated by stage-specific lncRNAs, circRNAs, and miRNAs were significantly enriched in epithelial differentiation and hair follicle morphogenesis. Regulatory network and gene co-expression analyses identified key transcripts controlling hair follicle development. We further predicted transcriptional factors (e.g. KLF4, LEF1, HOXC13, RBPJ, VDR, RARA, and STAT3) with stage-specific involvement in hair follicle morphogenesis. Through integrating these stage-specific genomic features with results from genome-wide association studies (GWAS) of five wool-related traits in 7135 Merino sheep, we detected developmental stages and genes that were relevant with wool-related traits in sheep. For instance, genes that were specifically upregulated at E105 were significantly associated with most of wool-related traits. A phenome-wide association study (PheWAS) demonstrated that candidate genes of wool-related traits (e.g. SPHK1, GHR, PPP1R27, CSRP2, EEF1A2, and PTPN1) in sheep were also significantly associated with dermatological, metabolic, and immune traits in humans.ConclusionsOur study provides novel insights into the molecular basis of hair follicle morphogenesis and will serve as a foundation to improve breeding for wool traits in sheep. It also indicates the importance of studying gene expression in the normal development of organs in understanding the genetic architecture of economically important traits in livestock. The datasets generated here are useful resources for functionally annotating the sheep genome, and for elucidating early skin development in mammals, including humans.

ISheep: an Integrated Resource for Sheep Genome, Variant and Phenotype.

iSheep: an Integrated Resource for Sheep Genome, Variant and Phenotype.

Biodiversity of Russian Local Sheep Breeds Based on Pattern of Runs of Homozygosity

Russian sheep breeds traditionally raised in specific environments are valuable parts of sociocultural heritage and economic component of the regions. However, the import of commercial breeds negatively influences the population sizes of local sheep populations and might lead to biodiversity loss. Estimation of the runs of homozygosity (ROH) in local sheep genomes is an informative tool to address their current genetic state. In this work, we aimed to address the ROH distribution and to estimate genome inbreeding based on SNP data to evaluate genetic diversity in Russian local sheep breeds. Materials for this study included SNP-genotypes from twenty-seven Russian local sheep breeds which were generated using the Illumina OvineSNP50 BeadChip (n = 391) or the Illumina Ovine Infinium HD BeadChip (n = 315). A consecutive runs method was used to calculate ROH which were estimated for each animal and then categorized in the ROH length classes. The ROH were found in all breeds. The mean ROH length varied from 86 to 280 Mb, while the ROH number ranged from 37 to 123. The genomic inbreeding coefficient varied from 0.033 to 0.106. Our findings provide evidence of low to moderate genomic inbreeding in major local sheep populations.

​Re-sequencing of Whole Genome Jordanian Awassi Ewes using Hiseq Sequencing Technology: Major Step Towards Sheep Genomic Selection

Background: Jordanian Awassi sheep (Ovis aries) is the dominant fat tail sheep breed that appeals to customers because of its various production systems, including fiber, meat and milk. This report is the first whole ewe genome sequence (WGS) of O. aries submitted in the NCBI database from Jordan. Methods: 64 Paired-end sequencing libraries were constructed and subjected to Illumina Hiseq 2500 sequencing system. High-quality reads were aligned against the reference sheep genome and detecting comprehensive sources (SNPs, InDels, SV, CNVs) of genetic variations. We have deposited data sequences at the NCBI under SRA (sequence reads archives) under the accession numbers SRR11128863, PRJNA574879. Result: Genome resequencing of Jordanian Awassi ewe was carried out with approximately 93.88 Gb with a mapping rate and effective mapping depths were 99.28% and 36.32. Around 19 million SNPs, 3,6 million InDels, 35,180 Structure variation and 13,524 copy number variation among the Jordanian ewe genome were detected. This wide range of genetic variation provides a framework for further genetic studies that will help understand the molecular basis underlying phenotypic variation of economically important traits in sheep and improve intrinsic defects in domestic sheep breeds.

Genome-Wide Detection of Copy Number Variations and Their Association With Distinct Phenotypes in the World's Sheep.

Copy number variations (CNVs) are a major source of structural variation in mammalian genomes. Here, we characterized the genome-wide CNV in 2059 sheep from 67 populations all over the world using the Ovine Infinium HD (600K) SNP BeadChip. We tested their associations with distinct phenotypic traits by conducting multiple independent genome-wide tests. In total, we detected 7547 unique CNVs and 18,152 CNV events in 1217 non-redundant CNV regions (CNVRs), covering 245 Mb (∼10%) of the whole sheep genome. We identified seven CNVRs with frequencies correlating to geographical origins and 107 CNVRs overlapping 53 known quantitative trait loci (QTLs). Gene ontology and pathway enrichment analyses of CNV-overlapping genes revealed their common involvement in energy metabolism, endocrine regulation, nervous system development, cell proliferation, immune, and reproduction. For the phenotypic traits, we detected significantly associated (adjusted P < 0.05) CNVRs harboring functional candidate genes, such as SBNO2 for polycerate; PPP1R11 and GABBR1 for tail weight; AKT1 for supernumerary nipple; CSRP1, WNT7B, HMX1, and FGFR3 for ear size; and NOS3 and FILIP1 in Wadi sheep; SNRPD3, KHDRBS2, and SDCCAG3 in Hu sheep; NOS3, BMP1, and SLC19A1 in Icelandic; CDK2 in Finnsheep; MICA in Romanov; and REEP4 in Texel sheep for litter size. These CNVs and associated genes are important markers for molecular breeding of sheep and other livestock species.

Immunogenetic variation at MHC class I loci in desert bighorn sheep (Ovis canadensis nelsoni) reflects natural and anthropogenic fragmentation

Abstract Desert bighorn sheep (Ovis canadensis nelsoni) are a charismatic ungulate native to western North America that live in mountainous regions. In the desert of the southwest United States, populations of bighorn sheep can be isolated by both natural and anthropogenic barriers. Isolation limits gene flow, exacerbates loss of genetic diversity to drift, and thus could affect immune responses to infectious agents. We sought to develop a relatively rapid method for determining MHC class I gene diversity in individual sheep. Both DNA and RNA were isolated from leukocytes collected from 154 adult bighorn sheep from across different mountain ranges in Southern California. Previously identified primers from domestic sheep were used to amplify exons 2 and 3 from OMHC I genes while appending adapters for PacBio circular consensus sequencing. We successfully identified over 40 unique MHC class I sequences expressed by bighorn sheep. Using a combination of phylogenic tree analysis and mapping MHC sequences to the domestic sheep genome, we detect at least 4 unique classical MHC class I regions. Comparison of results using either genomic DNA or mRNA (cDNA) as a template for analysis suggest that certain MHC alleles are expressed at different levels within leukocytes, while many potential pseudogenes were identified when genomic DNA was used as a template. Our data suggests using messenger RNA (cDNA) as a template to identify MHC class I diversity is superior to genomic DNA. Finally, we found clear patterns of MHC class I genotypes differentiated by population, which suggests that both natural and anthropogenic-induced population fragmentation can limit the diversity of MHC genes within a given population.

The pattern of runs of homozygosity and genomic inbreeding in world-wide sheep populations

Genome-wide pattern of runs of homozygosity (ROH) across ovine genome can provide a useful resource for studying diversity and demography history in sheep. We analyzed 50 k SNPs chip data of 2536 animals to identify pattern, distribution and level of ROHs in 68 global sheep populations. A total of 60,301 ROHs were detected in all breeds. The majority of the detected ROHs were <16 Mb and the average total number of ROHs per individual was 23.8 ± 13.8. The ROHs greater than 1 Mb covered on average 8.2% of the sheep autosomes, 1% of which was related to the ROHs with 1–4 Mb of length. The mean sum of ROH length in two-thirds of the populations was less than 250 Mb ranging from 21.7 to near 570 Mb. The level of genomic inbreeding was relatively low. The average of the inbreeding coefficients based on ROH (FROH) was 0.09 ± 0.05. It was rising in a stepwise manner with distance from Southwest Asia and maximum values were detected in North European breeds. A total of 465 ROH hotspots were detected in 25 different autosomes which partially surrounding 257 Refseq genes across the genome. Most of the detected genes were related to growth, body weight, meat production and quality, wool production and pigmentation. In conclusion, our analysis showed that the sheep genome, compared with other livestock species such as cattle and pig, displays low levels of homozygosity and appropriate genetic diversity for selection response and genetic merit gain.

Functional characterization of Copy Number Variations regions in Djallonké sheep.

A total of 184 Djallonké (West African Dwarf) sheep of Burkina Faso were analysed for Copy Number Variations (CNV) using Ovine 50K SNP BeadChip genotyping data and two different CNV calling platforms: PennCNV and QuantiSNP. Analyses allowed to identify a total of 63 candidate Copy Number Variations Regions (CNVR) on 11 different ovine chromosomes covering about 82.5Mb of the sheep genome. Gene-annotation enrichment analysis allowed to identify a total of 751 potential candidate ovine genes located in the candidate CNVR bounds. Functional annotation allowed to identify five statistically significant Functional Clusters (FC; enrichment factor >1.3) involving 61 candidate genes. All genes forming significantly enriched FC were located on ovine chromosome (OAR) 21. FC1 (22 genes including PAG4 and PAG6) and FC5 (three genes: CTSC, CTSW and CTSF), coding proteases (peptidases and cathepsins, respectively), were involved in reproductive performance and modulation of gestation. Both FC3 and FC4 were involved in inflammatory and immunologic response through coding serum amyloid A and B-box-type zinc finger proteins, respectively. Finally, FC2 consisted of 27 genes (including OR10G6 and OR8B8) involved in olfactory receptor activity, key for animals adapting to new food resources. CNVR identified on at least 15% of individuals were considered CNVR hotspots and further overlapped with previously reported quantitative trait loci (QTL). CNVR hotspots spanning genes putatively involved with lipid metabolism (SKP1, TCF7, JADE2, UBE2B and SAR1B) and differential expression in mammary gland (SEC24A and CDKN2AIPNL) on OAR5 and dairy traits (CCDC198 and SLC35F4) on OAR7 overlapped with QTL associated with lipid metabolism, milk protein yield and milk fat percentage. Information obtained from local sheep populations naturally adapted to harsh environments contributes to increase our understanding of the genomic importance of CNV.

Selective Sweeps Uncovering the Genetic Basis of Horn and Adaptability Traits on Fine-Wool Sheep in China.

Long-term natural and artificial selection leads to change in certain regions of the genome, resulting in selection signatures that can reveal genes associated with selected traits, such as horns (i.e., polled/horned), high-quality wool traits, and high-altitude hypoxia adaptability. These are complex traits determined by multiple genes, regulatory pathways, and environmental factors. A list of genes with considerable effects on horn and adaptability traits has not been found, although multiple quantitative trait loci (QTL) have been identified. Selection signatures could be identified using genetic differentiation (FST), polymorphism levels θπ, and Tajima’s D. This study aimed to identify selection signatures in fine-wool sheep and to investigate the genes annotated in these regions, as well as the biological pathways involved in horn and adaptability traits. For this purpose, the whole-genome sequence of 120 individuals from four breeds, which come from different elevations and habitats in China, was used to analyze selection signatures for horn and adaptability traits. Annotation of the consensus regions of FST and θπ ratios revealed a list of identified genes associated with polled/horned and high-altitude hypoxia adaptability traits, such as RXPF2, EERFC4, MSH6, PP1R12A, THBS1, ATP1B2, RYR2, and PLA2G2E. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified genes related primarily to mismatch repair, metabolism, vascular smooth muscle contraction, and cardiac muscle contraction. This is the first study to demonstrate that selection signatures play an important role in the polled/horned and high-altitude hypoxia adaptability traits of fine-wool sheep breeds that have undergone high-intensity selection and adapted to different ecological environments in China. Changes observed in the genome of fine-wool sheep may have acted on genomic regions that affect performance traits and provide a reference for genome design and breeding.

Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

BackgroundIntramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China’s most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12).ResultsWe identified a total of 26,247 genes and 6935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.ConclusionsOur study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

A global analysis of CNVs in Chinese indigenous fine-wool sheep populations using whole-genome resequencing

BackgroundCopy number variation (CNV) is an important source of genetic variation that has a significant influence on phenotypic diversity, economically important traits and the evolution of livestock species. In this study, the genome-wide CNV distribution characteristics of 32 fine-wool sheep from three breeds were analyzed using resequencing.ResultsA total of 1,747,604 CNVs were detected in this study, and 7228 CNV regions (CNVR) were obtained after merging overlapping CNVs; these regions accounted for 2.17% of the sheep reference genome. The average length of the CNVRs was 4307.17 bp. “Deletion” events took place more frequently than “duplication” or “both” events. The CNVRs obtained overlapped with previously reported sheep CNVRs to variable extents (4.39–55.46%). Functional enrichment analysis showed that the CNVR-harboring genes were mainly involved in sensory perception systems, nutrient metabolism processes, and growth and development processes. Furthermore, 1855 of the CNVRs were associated with 166 quantitative trait loci (QTL), including milk QTLs, carcass QTLs, and health-related QTLs, among others. In addition, the 32 fine-wool sheep were divided into horned and polled groups to analyze for the selective sweep of CNVRs, and it was found that the relaxin family peptide receptor 2 (RXFP2) gene was strongly influenced by selection.ConclusionsIn summary, we constructed a genomic CNV map for Chinese indigenous fine-wool sheep using resequencing, thereby providing a valuable genetic variation resource for sheep genome research, which will contribute to the study of complex traits in sheep.

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages.

Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.

93 A sheep model of sickle cell disease using CRISPR/Cas9 and somatic cell nuclear transfer

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, with more than 2 million people in the United States alone carrying the sickle gene. Approximately 100 000 of these people are homozygous and suffer from SCD. Worldwide, there are ∼4.4 million people with SCD. SCD is caused by a single A to T nucleotide replacement at the sixth codon of the β-globin gene, which results in the substitution of a valine for glutamate in the β-globin protein. This causes the resultant tetrameric haemoglobin molecule to be unstable and the red cells carrying this aberrant protein to “sickle,” decreasing the ability of these cells to carry oxygen. Sheep and humans exhibit a high degree of homology at the level of the genome. In addition, their anatomy, organ physiology, and immune system development closely parallel that of humans during fetal life. The ovine β-globin (HBB) gene shares 87.5% similarity with human HBB. Therefore, we hypothesised that the introduction of the “sickle” mutation in the sheep genome would lead to the SCD phenotype in sheep that could provide a valuable platform for evaluating prenatal and postnatal drug and gene therapies for this disease. In this study, we used a CRISPR/Cas9 gene-editing approach to introduce the SCD mutation into the sheep β-globin/HBB gene. We designed a single guide (sg)RNA targeting exon 1 of the sheep β-globin/HBB gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from IDT. Using the Lonza-4D-Nucleofector system, the Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs) along with 101-bp single-stranded oligodeoxynucleotides, flanking the sickle cell mutation to enable homology-directed repair. The transfected SFFs were then cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. After 2 days, DNA was extracted from one-third of the SFFs and the remainder were seeded individually into five 96-well plates by limited dilution. After 7 days of culture, individual colonies were expanded into 24-well plates and cultured for an additional 3 days. PCR-restriction fragment length polymorphism (RFLP) analysis using Image J software demonstrated a high rate of mutations (∼70%) by either indels or SCD mutation that led to the loss of the restriction enzyme site, which was further supported by the analysis of cell colonies. We isolated 59 single cell-derived SFF colonies and, based on PCR/RLFP assay, 31/59 (52%) of them contained biallelic mutations (either indels or point mutations) and were subsequently submitted for Sanger sequencing. The sequencing demonstrated that 3 colonies (9.6%) contained biallelic SCD mutations in the β-globin/HBB gene. These data demonstrate that we successfully introduced the SCD mutation into SFFs. These cells will be used in the production of the first large animal (sheep) SCD model by somatic cell nuclear transfer in fall of 2020. This research was supported by UAES project 1343 and by USDA/NIFA multistate research project W-4171.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P&amp;gt;0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.