Genome-wide detection of structural variation in some sheep breeds using whole-genome long-read sequencing data.

Genomic structural variants (SVs) constitute a significant proportion of genetic variation in the genome. The rapid development of long-reads sequencing has facilitated the detection of long-fragment SVs. There is no published study to detect SVs using long-read data from sheep. We applied a long-read mapping approach to detect SVs and characterized a total of 30,771 insertions, deletions, inversions and translocations. We identified 716, 916, 842 and 303 specific SVs in Southdown sheep, Alpine merino sheep, Qilian White Tibetan sheep and Oula sheep, respectively. We annotated these SVs and found that these SV-related genes were primarily enriched in the well-established pathways involved in the regulation of the immune system, growth and development and environmental adaptability. We detected and annotated SVs based on NGS resequencing data to validate the accuracy based on third-generation detection. Moreover, five candidate SVs were verified using the PCR method in 50 sheep. Our study is the first to use a long-reads sequencing approach to construct a novel structural variation map in sheep. We have completed a preliminary exploration of the potential effects of SVs on sheep.

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity.

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics ChromiumTM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (Apale and Adark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, Apale and Adark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.

Genetic Basis of Dorper Sheep (Ovis aries) Revealed by Long-Read De Novo Genome Assembly.

Dorper sheep (Ovis aries) (DPS), developed in the 1930s by crossing Dorset Horn and Blackhead Persian sheep in South Africa, is a world-famous composite breed for mutton production. The genetic basis underlying this breed is yet to be elucidated. Here, we report the sequencing and assembly of a highly contiguous Dorper sheep genome via integration of Oxford Nanopore Technology (ONT) sequencing and Hi-C (chromatin conformation capture) approaches. The assembled genome was around 2.64 Gb with a contig N50 of 73.33 Mb and 140 contigs in total. More than 99.5% of the assembled sequences could be anchored to 27 chromosomes and they were annotated with 20,450 protein-coding genes. Allele-specific expression (ASE) genes of Dorper sheep were revealed through ASE analysis and they were involved in the immune system, lipid metabolism, and environmental adaptation. A total of 5,701 and 456 allelic sites were observed in the SNP and indels loci identified from relevant whole-genome resequencing data. These allelic SNP and INDEL sites were annotated in 1,002 and 294 genes, respectively. Moreover, we calculated the number of variant sites and related genes derived from the maternal and paternal ancestors, revealing the genetic basis of outstanding phenotypic performance of Dorper sheep. In conclusion, this study reports the first reference genome of Dorper sheep and reveals its genetic basis through ASE. This study also provides a pipeline for mining genetic information of composite breeds, which has an implication for future hybrid-breeding practices.

Genome-Wide Selective Signatures Reveal Candidate Genes Associated with Hair Follicle Development and Wool Shedding in Sheep.

Hair follicle development and wool shedding in sheep are poorly understood. This study investigated the population structures and genetic differences between sheep with different wool types to identify candidate genes related to these traits. We used Illumina ovine SNP 50K chip genotyping data of 795 sheep populations comprising 27 breeds with two wool types, measuring the population differentiation index (Fst), nucleotide diversity (θπ ratio), and extended haplotype homozygosity among populations (XP-EHH) to detect the selective signatures of hair sheep and fine-wool sheep. The top 5% of the Fst and θπ ratio values, and values of XP-EHH < -2 were considered strongly selected SNP sites. Annotation showed that the PRX, SOX18, TGM3, and TCF3 genes related to hair follicle development and wool shedding were strongly selected. Our results indicated that these methods identified important genes related to hair follicle formation, epidermal differentiation, and hair follicle stem cell development, and provide a meaningful reference for further study on the molecular mechanisms of economically important traits in sheep.

Whole-genome re-sequencing association study on yearling wool traits in Chinese fine-wool sheep.

To investigate single nucleotide polymorphism (SNP) loci associated with yearling wool traits of fine-wool sheep for optimizing marker-assisted selection and dissection of the genetic architecture of wool traits, we conducted a genome-wide association study (GWAS) based on the fixed and random model circulating probability unification (FarmCPU) for yearling staple length (YSL), yearling mean fiber diameter (YFD), yearling greasy fleece weight (YGFW), and yearling clean fleece rate (YCFR) by using the whole-genome re-sequenced data (totaling 577 sheep) from the following four fine-wool sheep breeds in China: Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Qinghai fine-wool sheep (QHS), and Aohan fine-wool sheep (AHS). A total of 16 SNPs were detected above the genome-wise significant threshold (P = 5.45E-09), and 79 SNPs were located above the suggestive significance threshold (P = 5.00E-07) from the GWAS results. For YFD and YGFW traits, 7 and 9 SNPs reached the genome-wise significance thresholds, whereas 10 and 12 SNPs reached the suggestive significance threshold, respectively. For YSL and YCFR traits, none of the SNPs reached the genome-wise significance thresholds, whereas 57 SNPs exceeded the suggestive significance threshold. We recorded 14 genes located at the region of ±50-kb near the genome-wise significant SNPs and 59 genes located at the region of ±50-kb near the suggestive significant SNPs. Meanwhile, we used the Average Information Restricted Maximum likelihood algorithm (AI-REML) in the "HIBLUP" package to estimate the heritability and variance components of the four desired yearling wool traits. The estimated heritability values (h2) of YSL, YFD, YGFW, and YCFR were 0.6208, 0.7460, 0.6758, and 0.5559, respectively. We noted that the genetic parameters in this study can be used for fine-wool sheep breeding. The newly detected significant SNPs and the newly identified candidate genes in this study would enhance our understanding of yearling wool formation, and significant SNPs can be applied to genome selection in fine-wool sheep breeding.

Genome-wide association studies detects candidate genes for wool traits by re-sequencing in Chinese fine-wool sheep.

BACKGROUND: The quality and yield of wool determine the economic value of the fine-wool sheep. Therefore, discovering markers or genes relevant to wool traits is the cornerstone for the breeding of fine-wool sheep. In this study, we used the Illumina HiSeq X Ten platform to re-sequence 460 sheep belonging to four different fine-wool sheep breeds, namely, Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Aohan fine-wool sheep (AHS) and Qinghai fine-wool sheep (QHS). Eight wool traits, including fiber diameter (FD), fiber diameter coefficient of variance (FDCV), fiber diameter standard deviation (FDSD), staple length (SL), greasy fleece weight (GFW), clean wool rate (CWR), staple strength (SS) and staple elongation (SE) were examined. A genome-wide association study (GWAS) was performed to detect the candidate genes for the eight wool traits. RESULTS: A total of 8.222 Tb of raw data was generated, with an average of approximately 8.59X sequencing depth. After quality control, 12,561,225 SNPs were available for analysis. And a total of 57 genome-wide significant SNPs and 30 candidate genes were detected for the desired wool traits. Among them, 7 SNPs and 6 genes are related to wool fineness indicators (FD, FDCV and FDSD), 10 SNPs and 7 genes are related to staple length, 13 SNPs and 7 genes are related to wool production indicators (GFW and CWR), 27 SNPs and 10 genes associated with staple elongation. Among these candidate genes, UBE2E3 and RHPN2 associated with fiber diameter, were found to play an important role in keratinocyte differentiation and cell proliferation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results, revealed that multitude significant pathways are related to keratin and cell proliferation and differentiation, such as positive regulation of canonical Wnt signaling pathway (GO:0090263). CONCLUSION: This is the first GWAS on the wool traits by using re-sequencing data in Chinese fine-wool sheep. The newly detected significant SNPs in this study can be used in genome-selective breeding for the fine-wool sheep. And the new candidate genes would provide a good theoretical basis for the fine-wool sheep breeding.

The complete mitochondrial genome sequence and phylogenetic analysis of Alpine Merino sheep (Ovis aries).

Alpine Merino sheep is one of the most important fine-wool sheep breeds in China. In this study, we present the complete mitogenome of Alpine Merino sheep for the first time. The genome has a length of 16,619bp, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (D-loop). Phylogenetically, the Alpine Merino sheep is closer to Oula Tibetan sheep and Tashkurgan sheep. This report provided new data for the phylogeny of Alpine Merino sheep.

Genomic Prediction of Additive and Dominant Effects on Wool and Blood Traits in Alpine Merino Sheep.

Dominant genetic effects may provide a critical contribution to the total genetic variation of quantitative and complex traits. However, investigations of genome-wide markers to study the genomic prediction (GP) and genetic mechanisms of complex traits generally ignore dominant genetic effects. The increasing availability of genomic datasets and the potential benefits of the inclusion of non-additive genetic effects in GP have recently renewed attention to incorporation of these effects in genomic prediction models. In the present study, data from 498 genotyped Alpine Merino sheep were adopted to estimate the additive and dominant genetic effects of 9 wool and blood traits via two linear models: (1) an additive effect model (MAG) and (2) a model that included both additive and dominant genetic effects (MADG). Moreover, a method of 5-fold cross validation was used to evaluate the capability of GP in the two different models. The results of variance component estimates for each trait suggested that for fleece extension rate (73%), red blood cell count (28%), and hematocrit (25%), a large component of phenotypic variation was explained by dominant genetic effects. The results of cross validation demonstrated that the MADG model, comprising additive and dominant genetic effects, did not display an apparent advantage over the MAG model that included only additive genetic effects, i.e., the model that included dominant genetic effects did not improve the capability for prediction of the genomic model. Consequently, inclusion of dominant effects in the GP model may not be beneficial for wool and blood traits in the population of Alpine Merino sheep.

Genome-Wide Association Study Using Individual Single-Nucleotide Polymorphisms and Haplotypes for Erythrocyte Traits in Alpine Merino Sheep.

Adaptation to high-altitude hypoxia is essential for domestic animals, such as yak, Tibetan chicken, and Tibetan sheep, living on high plateaus, as it ensures efficient oxygen absorption and utilization. Red blood cells are the primary medium for transporting oxygen in the blood. However, little is known about the genetic mechanism of erythrocyte traits. Genome-wide association studies (GWASs) based on single markers or haplotypes have identified potential mechanisms for genetic variation and quantitative traits. To identify loci associated with erythrocyte traits, we performed a GWAS based on the method of the single marker and haplotype in 498 Alpine Merino sheep for six erythrocyte traits: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC volume distribution width coefficient of variation (RWD_CV). Forty-two significant single-nucleotide polymorphisms (SNPs) associated with the six erythrocyte traits were detected by means of a single-marker GWAS, and 34 significant haplotypes associated with five erythrocyte traits were detected by means of haplotype analysis. We identified six genes (DHCR24, SPATA9, FLI1, PLCB1, EFNB2, and SH2B3) as potential genes of interest via gene function annotations, location, and expression variation. In particular, FLI1 and PLCB1 were associated with hematopoiesis and erythropoiesis, respectively. These results provide a theoretical basis for analyzing erythrocyte traits and high-altitude hypoxia adaptation in Alpine Merino sheep and will be a useful reference for future studies of plateau-dwelling livestock.

A case report regarding general anesthesia management of a patient with pulmonary vein stenosis that underwent balloon dilatation and stent implantation.

RATIONALE: Pulmonary vein stenosis (PVS) is a rare cardiovascular deformity that can lead to high mortality if left untreated. Patients frequently experience multiple complications such as hemoptysis, pulmonary hypertension, bronchial venous rupture and cardiac insufficiency. Currently, pulmonary vein stenosis balloon dilatation (stent implantation) is the only treatment, and this can be performed under local or general anesthesia. However, a case report on the general anesthesia management of PVS has not been previously reported. In this case report, we discuss anesthetic considerations in patients with PVS, focusing specifically on perioperative airway and circulatory management as well as the risk evaluation, and the appropriate effective management of all potential complications intraoperatively. PATIENT CONCERNS: A 58-year-old male patient was admitted because of coughing rusty sputum for during 2 years as well as experiencing dyspnea and chest distress after exertion or exercising. The difficulty breathing and chest distress had been going on for a year. This patient had undergone circumferential pulmonary vein isolation twice during the last 2 years. DIAGNOSES: Based on the transthoracic echocardiography and computed tomography, this patient's diagnosis was considered as pulmonary vein stenosis, pulmonary hypertension (secondary), and pulmonary arteriovenous thrombosis. INTERVENTIONS: We considered that such severe patients with PVS require respiratory and circulatory supports and perhaps emergency surgical interventions. Henceforth, we administered general anesthesia to the patient and had extracorporeal membrane oxygenation (ECMO) on standby. OUTCOMES: The duration of the surgery was approximately 4 hours, the intraoperative vital signs were stable, no pericardial effusion was observed postoperatively, the blood flow in the pulmonary vein stent was smooth, and the patient was discharged 7 days later following rehabilitation. LESSONS: This surgical procedure involving respiratory and circulatory supports requires the involvement of different medical personnel such as interventionists, anesthesiologists, and surgeons. Therefore, multidisciplinary cooperation under general anesthesia will undoubtedly benefit such patients.