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.

circUBE3C modulates myoblast development by binding to miR-191 and upregulating the expression of p27.

Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.

Genetic variant of the sheep E2F8 gene and its associations with litter size.

The economic efficiency of sheep breeding, aiming to enhance productivity, is a focal point for improvement of sheep breeding. Recent studies highlight the involvement of the Early Region 2 Binding Factor transcription factor 8 (E2F8) gene in female reproduction. Our group's recent genome-wide association study (GWAS) emphasizes the potential impact of the E2F8 gene on prolificacy traits in Australian White sheep (AUW). Herein, the purpose of this study was to assess the correlation of the E2F8 gene with litter size in AUW sheep breed. This work encompassed 659 AUW sheep, subject to genotyping through PCR-based genotyping technology. Furthermore, the results of PCR-based genotyping showed significant associations between the P1-del-32bp bp InDel and the fourth and fifth parities litter size in AUW sheep; the litter size of those with genotype ID were superior compared to those with DD and II genotypes. Thus, these results indicate that the P1-del-32bp InDel within the E2F8 gene can be useful in marker-assisted selection (MAS) in sheep.

Copy number variations within fibroblast growth factor 13 gene influence growth traits and alternative splicing in cattle.

Previous researches revealed a copy number variation (CNV) region in the bovine fibroblast growth factor 13 (FGF13) gene. However, its effects remain unknown. This study detected the various copy number types in seven Chinese cattle breeds and analysed their population genetic characteristics and effects on growth traits and transcription levels. Copy number Loss was more frequent in Caoyuan Red cattle and Xianan cattle than in the other breeds. Association analysis between CNV and growth traits of Qinchuan indicated that the CNV was significantly related to chest depth, hip width and hucklebone width (P < 0.05). Additionally, the growth traits of individuals with copy number Loss were significantly inferior to those with copy number Gain or Median (P < 0.05). Besides, we found two splicing isoforms, AS1 and AS2, in FGF13 gene, which resulted from alternative 5' splicing sites of intron 1. These isoforms showed varied expression levels in various tissues. Moreover, CNV was significantly and negatively associated with the mRNA expression of AS1 (r = -0.525, P < 0.05). The CNVs in bovine FGF13 gene negatively regulated growth traits and gene transcription. These observations provide new insights into bovine FGF13 gene, delivering potentially useful information for future Chinese cattle breeding programs.

Self-Assembling Nanoparticle Hemagglutinin Influenza Vaccines Induce High Antibody Response.

As a highly pathogenic avian virus, H5 influenza poses a serious threat to livestock, the poultry industry, and public health security. Hemagglutinin (HA) is both the dominant epitope and the main target of influenza-neutralizing antibodies. Here, we designed a nanoparticle hemagglutinin influenza vaccine to improve the immunogenicity of the influenza vaccine. In this study, HA5 subtype influenza virus was used as the candidate antigen and was combined with the artificially designed double-branch scaffold protein I53_dn5 A and B. A structurally correct and bioactive trimer HA5-I53_dn5B/Y98F was obtained through secretion and purification using an insect baculovirus expression system; I53_dn5A was obtained by purification using a prokaryotic expression system. HA5-I53_dn5B/Y98F and I53_dn5A self-assembled into spherical nanoparticles (HA5-I53_dn5) in vitro with a diameter of about 45 nm. Immunization and serum test results showed that both HA5-I53_dn5B/Y98F and HA5-I53_dn5 could induce HA5-specific antibodies; however, the immunogenicity of HA5-I53_dn5 was better than that of HA5-I53_dn5B/Y98F. Groups treated with HA5-I53_dn5B and HA5-I53_dn5 nanoparticles produced IgG antibody titers that were not statistically different from those of the nanoparticle-containing adjuvant group. This production of trimerized HA5-I53_dn5B and HA5-I53_dn5 nanoparticles using baculovirus expression provides a reference for the development of novel, safe, and efficient influenza vaccines.

Transcriptome Analysis of Compensatory Growth and Meat Quality Alteration after Varied Restricted Feeding Conditions in Beef Cattle.

Compensatory growth (CG) is a physiological response that accelerates growth following a period of nutrient limitation, with the potential to improve growth efficiency and meat quality in cattle. However, the underlying molecular mechanisms remain poorly understood. In this study, 60 Huaxi cattle were divided into one ad libitum feeding (ALF) group and two restricted feeding groups (75% restricted, RF75; 50% restricted, RF50) undergoing a short-term restriction period followed by evaluation of CG. Detailed comparisons of growth performance during the experimental period, as well as carcass and meat quality traits, were conducted, complemented by a comprehensive transcriptome analysis of the longissimus dorsi muscle using differential expression analysis, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and weighted correlation network analysis (WGCNA). The results showed that irrespective of the restriction degree, the restricted animals exhibited CG, achieving final body weights comparable to the ALF group. Compensating animals showed differences in meat quality traits, such as pH, cooking loss, and fat content, compared to the ALF group. Transcriptomic analysis revealed 57 genes and 31 pathways differentially regulated during CG, covering immune response, acid-lipid metabolism, and protein synthesis. Notably, complement-coagulation-fibrinolytic system synergy was identified as potentially responsible for meat quality optimization in RF75. This study provides novel and valuable genetic insights into the regulatory mechanisms of CG in beef cattle.

Comprehensive comparison of two types of algorithm for circRNA detection from short-read RNA-Seq.

MOTIVATION: Circular RNA is generally formed by the 'back-splicing' process between the upstream splice acceptor and the downstream donor in/not in the regulation of the corresponding RNA-binding proteins or cis-elements. Therefore, more and more software packages have been developed and they are mostly based on the identification of the back-spliced junction reads. However, recent studies developed two software tools that can detect circRNA candidates by constructing k-mer table or/and de Bruijn graph rather than reads mapping. RESULTS: Here, we compared the precision, sensitivity and detection efficiency between software tools based on different algorithms. Eleven representative detection tools with two types of algorithm were selected for the overall pipeline analysis of RNA-seq datasets with/without RNase R treatment in two cell lines. Precision, sensitivity, AUC, F1 score and detection efficiency metrics were assessed to compare the prediction tools. Meanwhile, the sensitivity and distribution of highly expressed circRNAs before and after RNase R treatment were also revealed by their enrichment, unaffected and depleted candidate frequencies. Eventually, we found that compared to the k-mer based tools, CIRI2 and KNIFE based on reads mapping had relatively superior and more balanced detection performance regardless of the cell line or RNase R (-/+) datasets. AVAILABILITY AND IMPLEMENTATION: All predicted results and source codes can be retrieved from https://github.com/luffy563/circRNA_tools_comparison. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The InDel variants of sheep IGF2BP1 gene are associated with growth traits.

Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) plays positive roles in the growth, proliferation of cells and early embryos development by binding mRNA targets. Recently, it had been shown that some polymorphic loci within IGF2BP1 gene were associated with growth traits in animals, especially in goats. Therefore, it has been hypothesized that some variants within IGF2BP1 gene may be also involved in growth traits of sheep. Nine insertion/deletion (InDel) mutations within IGF2BP1 were identified and three loci were polymorphic. Meanwhile, the association analyses between three InDels and growth traits were carried out in 745 sheep. The results showed that all InDels included 5 bp InDel in downstream region, 9 bp InDel in intron 4 and 15 bp InDel in intron 2 within IGF2BP1 were significantly associated with growth traits (p<.05). Furthermore, at 5 and 9 bp InDel loci, the individuals of heterozygous genotype (ID) had superior growing performance especially at body weight (BW). In all, three InDels were crucial variants correlated with growth traits and could be applied in marker-assisted selection (MAS) in sheep.

Survey of the relationship between polymorphisms within the BMPR1B gene and sheep reproductive traits.

The BMPRIB gene is one of the main genes that can be used as a molecular genetic marker for the early selection of highly productive ewes. It is well-documented that the p.Q249R (g.746A > G) is the first mutation in the kinase domain of the BMPR1B gene that is highly related to increased ovulation rate and litter size. It is likely that the presence of the p.Q249R mutation in the sheep population is one of the factors contributing to the outstanding productivity of the sheep. Moreover, in recent years, researchers have been explored other polymorphisms in the BMPR1B gene with respect to reproductive traits in sheep. Therefore, we carried out the current study to evaluate the association between polymorphisms in this gene and sheep litter size from all appropriate studies. As a result, among 41 polymorphisms in the ovine BMPRIB gene, eight variants, including p.Q249R (g.746A > G), g.29362047T > C, g.29427689G > A, BMPR1B-2 (ss:1960972599), g.29382337G > A, g.29382340G > A, rs1092293287 (10 bp insertion/deletion) and g.29380965A > G were found to be associated with litter size in sheep. This systematic analysis presents the most current data evidence for BMPRIB polymorphisms, highlighting the need for further large-scale studies to determine more important variants.

Screen of small fragment mutations within the sheep thyroid stimulating hormone receptor gene associated with litter size.

The thyroid stimulating hormone receptor (TSHR), a glycoprotein hormone receptor, plays an important role in metabolic regulation and photoperiod control in the time of reproduction in birds and mammals. Previous genome-wide association studies revealed that the TSHR gene was related to reproduction and its function was identified in female reproduction, but rare studies reported the polymorphism of TSHR gene. However, the molecular mutations of the TSHR gene in sheep have not been reported so far. Herein, we explored potential polymorphisms of the sheep TSHR gene, and a 29 bp nucleotide sequence variant (rs1089565492) was identified in the AUW sheep. There were three genotypes of the 29 bp variant locus detected which named 'II' 'DD' and 'ID' been identified. Association analysis results showed the 29 bp variant was significantly associated with the litter size of the AUW sheep (p < 0.05). This finding suggests that the 29 bp nucleotide sequence variant within TSHR gene could be a candidate marker of reproduction traits for sheep breeding improving through the marker-assisted selection (MAS).

Investigation of Genetic Effects of Nucleotide Variants Within the Goat PRNT Gene on Growth Performance.

Our previous study has firstly pointed that three nucleotide variants (g.-11C > T, g.117A > G, and g.149C > T) of the goat PRNT gene can significantly influence litter size. Given litter size is positively correlated with growth performance, we consider whether the PRNT gene also acts on the growth performance in goats. In this work, a correlation analysis among different litter size types and growth traits of Shaanbei white cashmere (SBWC) goats was performed, and results showed that a positive correlation did exist in our detected population (P < 0.01). Then, the association among different genotypes of three variations and goat growth performance was measured. Our results pointed to g.117A > G being significantly associated with the cannon circumference (P = 4.60E-05) while no significant effect was found between another two SNPs and growth traits after the Bonferroni's correction (P*n < 0.05). Together, this is the first report about the influence of the PRNT gene on the growth of goat and g.117A > G can be regarded as a possible DNA marker applying for MAS breeding.

A deletion mutation within the goat AKAP13 gene is significantly associated with litter size.

A-kinase anchoring protein 13 (AKAP13) is one of the AKAP protein family members, which is correlated with estrogen receptors (ERs) and progesterone receptor (PR) activity. Consequently, the AKAP13 gene is considered to be one of the candidate genes for regulating female fertility. Hence, the objectives of this study were to discover the potential insertion/deletion (indel) variants within the AKAP13 gene and evaluate their associations with litter size of Shaanbei white cashmere goats (SBWC) to screen candidate genes for the molecular marker-assisted selection (MAS). Ultimately, we found the 16-bp deletion of AKAP13 gene which displayed three genotypes (II, ID and DD). However, it was not confirmed to Hardy-Weinberg equilibrium (HWE) in the tested population. Statistical analysis demonstrated that this 16-bp indel locus was significantly associated with litter size in goats (p < 0.05), in which the ID genotype was a key genotype for increasing litter size in goats. Besides, independent χ2 tests between different genotypes and litter size showed that high-prolific groups had higher frequency of the 'D' allele (p < 0.05). Briefly, AKAP13 gene is a candidate gene for improving fertility, and its 16-bp indel locus can be used as a valid DNA molecular marker for the MAS in goat breeding.

Insertion/deletion (Indel) variant of the goat RORA gene is associated with growth traits.

RAR related orphan receptor A (RORA), which encodes the retinoid-acid-related orphan receptor alpha (RORα), is a clock gene found in skeletal muscle. Several studies have shown that RORα plays an important role in bone formation, suggesting that RORA gene may take part in the regulation of growth and development. The purpose of this research is to study the insertion/deletion (indel) variations of the RORA gene and investigate the relationship with the growth traits of Shaanbei white cashmere (SBWC) goats. Herein, the current study identified that the P4-11-bp and P11-28-bp deletion sites are polymorphic among 12 pairs of primers within the RORA gene in the SBWC goats (n = 641). Moreover, the P11-28-bp deletion locus was significantly related to the body height (p = 0.046), height at hip cross (p = 0.012), and body length (p = 0.003). Both of P4-11-bp and P11-28-bp indels showed the moderate genetic diversity (0.25

Investigation on mRNA expression and genetic variation within goat SMAD2 gene and its association with litter size.

The SMAD family member 2 (SMAD2), a member of the TGF-beta superfamily, executes a significant part in the oogenesis and ovulation process. A genome-wide selective sweep analysis also found SMAD2 was different in the fertility groups of Laoshan dairy goats; whether this gene was linked to litter size was unknown. Therefore, SMAD2 was chosen to study its effects on Shaanbei white cashmere goat reproduction and mRNA expression profile. Herein, the mRNA expression level of SMAD2 was firstly determined in female goat tissues, revealing significant differences in mRNA levels of different tissues (p < 0.05), including ovary tissue, indicating a potential role for SMAD2 in goat prolificacy. Then, using six pairs of primers, only one indel locus (P3-Del-12-bp) was found to be polymorphic in goat SMAD2 (n = 501). ANOVA also revealed that a P3-Del-12-bp deletion was significantly related to first-born litter size (p = 0.037). The Chi-square (χ2) test revealed that the ID genotype was significantly more prevalent in mothers with multiple lambs (p = 0.01), indicating that heterozygous individuals (ID) are more likely to produce multiple lambs. Our findings suggest that the SMAD2 gene's P3-Del-12-bp deletion could be used to improve goat breeds by assisting with litter size selection.

Investigation of the association between goat DNMT3B gene polymorphism and growth traits.

The DNA methyltransferase 3 beta (DNMT3B) gene is key for DNA methylation and has been well recognized in regulating growth and development. A previous observation indicated that an 11-bp indel of DNMT3B affected the reproductive traits in goats, yet the effect of this polymorphism on body measurement traits in goats has not been reported. This study aims to investigate the associations between DNMT3B gene polymorphism and goat growth traits. We investigated this 11-bp indel in 2184 goats and three genotypes have been found in Shaanbei white cashmere goat (SBWC): insertion/insertion (II), deletion/deletion (DD) and insertion/deletion (ID). Only ID and DD genotypes were detected in Nubian goats and Guizhou heima goat (GZHM). The allele frequencies analyzed revealed that the 'D' allele frequencies were higher in all three goat breeds. Further association analysis demonstrated that this indel is markedly associated with the cannon circumference (CC) and cannon circumference index (CCI) of SBWC and cannon circumference (CC) of Nubian goats (p < .05). The CC and CCI are essential indicators to measure the growth status of goats. In summary, our study sheds some light on the potential impact of the 11-bp indel polymorphism of the DNMT3B gene on improving the growth traits in goats.

Goat CLSTN2 gene: tissue expression profile, genetic variation, and its associations with litter size.

Calsyntenin-2 (CLSTN2) is involved in cell proliferation, differentiation, cell death, tumorigenesis, and follicular expression. Although CLSTN2 has been identified as a potential candidate gene for sheep prolificacy, no studies have been done on its effect on goat prolificacy. The purpose of this study was to identify mRNA expression and genetic variation within goat CLSTN2, and its association with prolificacy. Herein, we uncovered significant differences in mRNA levels of the CLSTN2 gene in different tissues in female goats (p < 0.01), including ovary tissue. Nine putative indels were designed to investigate their correlation to litter size, but only one 16-bp deletion was discovered in female Shaanbei white cashmere goats (n = 902). We discovered that a 16-bp deletion within the CLSTN2 gene was significantly correlated with first-born litter size (p = 0.0001). As shown by the chi-squared test, the genotypic II of single-lambs and multi-lambs was dramatically higher than with genotype ID (p = 0.005). Our findings suggest that indel within the CLSTN2 gene is a candidate gene affecting prolificacy in goats and may be used for Marker Assisted Selection (MAS) in goats.

A 14-bp deletion in bovine EPAS1 gene is associated with carcass traits.

EPAS1 (Endothelial PAS Domain Protein 1) gene is well-known for its function in plateau hypoxia adaptability. It encodes HIF-2α, which involved in the induction of genes regulated by oxygen and then affects multiple physiological processes such as angiogenesis and energy metabolism. All of these indicate it may affect the development of animals. In this study, a 14-bp deletion in EPAS1 gene was uncovered in Shandong black cattle population (n = 502). Two genotypes (II and ID) were found and the frequency of the homozygous II genotype is higher than the heterozygous ID genotype. This population is consisted with HWE (p > 0.05). And more importantly, the 14-bp deletion was associated with outside flat (p = 0.003), brisket (p = 0.001), and knuckle (p = 0.032). These findings suggested that the 14-bp deletion is significantly associated with carcass traits, which could be served as a molecular marker applied to cow breeding.

Copy number variations of the KAT6A gene are associated with body measurements of Chinese sheep breeds.

Copy number variation (CNV) is one kind of genomic structure variations and presents as gains and losses of genomic fragments. More recently, we have made an atlas of CNV maps for livestock. In the future, it is a primary focus to determine the phenotypic effects of candidate CNVs. Lysine Acetyltransferase 6 A (KAT6A) is a protein coding gene and plays a critical role in many cellular processes. However, the effects of KAT6A CNVs on sheep body measurements remains unknown. In this study, we performed quantitative polymerase chain reaction (qPCR) to detect the presences and distributions of three CNV regions within KAT6A gene in 672 sheep from four Chinese breeds. Association analysis indicated that the three CNVs of KAT6A gene were significantly associated with body measurement(s) in Small-tailed Han sheep (STH) and Hu sheep (HU) (p < 0.05), while no effects on Large-tailed Han sheep (LTH) were observed (p > 0.05) were observed. Additionally, only one CNV was significantly associated with body measurement (body length) in Chaka sheep (CK) (p < 0.05). Our study provided evidence that the CNV(s) of KAT6A gene could be used as candidate marker(s) for molecular breedings of STH, HU, and CK breeds.

Distribution of DGAT1 copy number variation in Chinese goats and its associations with milk production traits.

Goat is an important sector for meat and dairy products. Diacylglycerol O-acyltransferase 1 (DGAT1), which is a key gene in milk production, has been recently detected to overlap with a novel copy number variation (CNV) in goats. CNVs could be genetic markers providing new insights into the genetic basis of phenotypic variation. Up to now, there are no reports on the DGAT1-related CNV (DGAT1 CNV) in Chinese goats. This study first detected the distribution of the DGAT1 CNV in Chinese seven goat breeds, finding substantial differences among dairy, meat, and fiber goats (P < 0.01). The association analysis between the DGAT1 CNV and milk production traits revealed significant associations: Xinong Sannen (XS) dairy goat with copy number loss type had higher freezing point depression (FPD) (P < 0.01) and milk solids-not-fat (SNF) content (P < 0.05). Overall, our study unraveled the distribution of DGAT1 CNV in Chinese goats for the first time and found the potential role of this CNV in the marker-assisted selection of dairy goat breeding.

Insertion/deletions within the bovine FoxO1 gene and their association analysis with growth traits in three Chinese cattle breeds.

FOXO1 (FKHR) gene, as a transcription factor, plays a vital role in animal growth and development, participating in many biological processes. The aim of this study was to ascertain Insertion/deletions (Indels) polymorphism within bovine FoxO1 gene in 679 Chinese adult cows and associate them with stature traits. Two Indels (named as Indel-3 and Indel-4, recorded as rs383545622 and rs525318770 in NCBI, respectively) were successfully genotyped by the Once PCR method, which was reliable, rapid and cost effective for simultaneous detection of two or more Indels. Indel-3 and Indel-4 were located at the second intron. All four different haplotypes (H1: D3D4, H2: I3D4, H3: D3I4, H4: I3I4) could be identified, and the D (del-) allele, DD (del-/del-) genotype and D3D4 haplotype retained the highest frequency. However, individuals with DI (D3I3, D4I4 or H1H4/H2H3 genotype) showed significantly better phenotypic traits than those with the other genotypes in Nanyang cattle, showing a hybrid vigor. The results implied that this DI genotype can be applied to early selective breeding to improve the productivity of Nanyang cattle. Our results suggested that these two Indels within the bovine FoxO1 gene might be used as genetic markers for marker-assisted selection (MAS) in cattle breeding and genetics.