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In order to investigate the effect of FecB on litter size and growth and development traits of Suhu meat sheep and the inheritance patterns of FecB between parents and offspring in the population. In this experiment, 2241 sheep from the Suhu meat sheep population were tested for FecB using capillary electrophoresis. We combined the lambing records of 473 ewes, the growth trait records of 881 sheep at both the birth and weaning (2-month-old) stages, and the complete genealogical records of 643 lambs to analysis the distribution of FecB in the Suhu meat sheep breeding population, its effect on litter size of ewes, growth and development of lambs, and the inheritance patterns of FecB. The results showed that there were three genotypes of FecB in the Suhu meat sheep population, namely the AA genotype, AG genotype, and GG genotype. FecB in this population has a moderate polymorphism (0.25 < PIC < 0.5), and deviates from Hardy-Weinberg disequilibrium (p < 0.05). The litter size of GG genotype ewes was significantly higher than that with the AG and AA genotypes ( p < 0.01). A Chi-square test showed that the inheritance patterns of FecB follows Mendel's Laws of Inheritance ( p > 0.05). An association analysis of different genotypes of FecB with body weight and body size of Suhu meat sheep at birth and weaning revealed that FecB adversely affects the early growth and development of Suhu meat sheep. In summary, FecB can improve the litter size of ewes but it has negative effects on the early growth and survival rate of lambs in sheep. Therefore, FecB test results and feeding management measures should be comprehensively applied to improve the reproductive performance of ewes, the survival rate and production performance of lambs in sheep production, and thus improve the economic benefits of sheep farms.

Previous studies have demonstrated that CUX1 could contribute to the proliferation of DPCs in vitro, but the upstream transcriptional regulatory mechanisms of CUX1 remain largely unknown. This study aimed to investigate the upstream transcriptional regulators of CUX1 to enhance our comprehension of the mechanism of action of the CUX1 gene in ovine DPCs. Initially, the JASPAR (2024) software was used to predict the upstream target transcription factors for the CUX1 gene. Subsequently, through RT-qPCR and a double luciferase reporter assay, the interaction between SP1 , KROX20 , and CUX1 was established, respectively. The results indicated that SP1 and KROX20 were two highly reliable upstream transcription regulators for the CUX1 gene. Additionally, we found that SP1 promoted the proliferation of DPCs by overexpressing SP1 in DPCs, and KROX20 inhibited the proliferation of DPCs by overexpressing KROX20 in DPCs. These findings are also consistent with the transcriptional regulation of CUX1 by SP1 and KROX20 , respectively. This study suggests that the effect of DPC proliferation in vitro by CUX1 may regulated by the transcription factors SP1 and KROX20 .

This study aims to analyze the whole genome sequencing of E. coli F17 in antagonistic and susceptible Hu sheep lambs. The objective is to investigate the critical mutation loci in sheep and understand the genetic mechanism of sheep resistance to E. coli F17 at the genome level. Antagonist and susceptible venous blood samples were collected from Hu sheep lambs for whole genome sequencing and whole genome association analysis. A total of 466 genes with significant SNPs ( p < 1.0 × 10 -3 ) were found. GO and KEGG enrichment analysis and protein interaction network analysis were performed on these genes, and preliminary investigations showed that SNPs on CTNNB1, CDH8, APOD, HCLS1, Tet2, MTSS1 and YAP1 genes may be associated with the antagonism and susceptibility of Hu sheep lambs to E. coli F17. There are still some shortcomings that have not been explored via in vivo and in vitro functional experiments of the candidate genes, which will be our next research work. This study provides genetic loci and candidate genes for resistance of Hu sheep lambs to E. coli F17 infection, and provides a genetic basis for breeding disease-resistant sheep.

Previous studies have shown that melatonin has a certain regulatory effect on the growth of sheep wool. However, the mechanism of melatonin action remains unknown. In the present study, we aimed to understand the role of exogenous melatonin in the dermal papilla cells of Hu sheep. To confirm the optimal melatonin treatment regimen for Hu sheep dermal papilla cells, we detected the cell viability by exposing them to different concentrations of melatonin and different treatment times. The results showed that cell viability was best when dermal papilla cells were treated with 1000 pg/ml of melatonin for 48 h. According to the results of qPCR, CCK-8, EDU, Western blot, and Flow cytometry analysis, we found that 1000 pg/ml melatonin promoted the proliferation and inhibited the apoptosis of dermal papilla cells compared with the exogenous melatonin blank group (control group). Furthermore, we also found that 1000 pg/ml of melatonin promoted the cell cycle progress of dermal papilla cells according to the results of qPCR and Flow cytometry analysis. Overall, our findings showed that melatonin plays an important role in the dermal papilla cells of Hu sheep.

Dorper and Hu sheep exhibit different characteristics in terms of reproduction, growth, and meat quality. Comparison of the genomes of two breeds help to reveal important genomic information. In this study, whole genome resequencing of 30 individuals (Dorper, DB and Hu sheep, HY) identified 15,108,125 single nucleotide polymorphisms (SNPs). Population differentiation ( Fst ) and cross population extended haplotype homozygosity (XP-EHH) were performed for selective signal analysis. In total, 106 and 515 overlapped genes were present in both the Fst results and XP-EHH results in HY vs DB and in DB vs HY, respectively. In HY vs DB, 106 genes were enriched in 12 GO terms and 83 KEGG pathways, such as ATP binding (GO:0005524) and PI3K-Akt signaling pathway (oas04151). In DB vs HY, 515 genes were enriched in 109 GO terms and 215 KEGG pathways, such as skeletal muscle cell differentiation (GO:0035914) and MAPK signaling pathway (oas04010). According to the annotation results, we identified a series of candidate genes associated with reproduction ( UNC5C , BMPR1B , and GLIS1 ), meat quality ( MECOM , MEF2C , and MYF6 ), and immunity ( GMDS , GALK1 , and ITGB4 ). Our investigation has uncovered genomic information for important traits in sheep and provided a basis for subsequent studies of related traits.

This study aimed to understand the expression level of YAP1 in the skeletal muscle of Hu sheep and to reveal the regulatory mechanism of YAP1 on Hu sheep skeletal muscle satellite cells (SMSCs). Previous research by our group has found that YAP1 may affect the growth and development of Hu sheep skeletal muscle. In the present study, we found the expression of YAP1 in the skeletal muscle is higher than in other tissues of Hu sheep. Then, we detected the effect of YAP1 on proliferation and differentiation in Hu sheep SMSCs. According to the results of qPCR, CCK-8, EDU, and Western blot, compared to the group of negative control, overexpression of YAP1 promoted the proliferation and inhibited the differentiation of SMSCs according to the results of qPCR, CCK-8, EDU, Western blot, while the interference of YAP1 was on the contrary. Overall, our study suggests that YAP1 is an important functional molecule in the growth and development of skeletal muscle by regulating the proliferation and differentiation of SMSCs. These findings are of great use for understanding the roles of YAP1 in the skeletal muscle of Hu sheep.

This study aimed to identify the target genes of IGFBP3（insulin growth factor binding protein）protein and to investigate its target genes effects on the proliferation and differentiation of Hu sheep skeletal muscle cells. IGFBP3 was an RNA-binding protein that regulates mRNA stability. Previous studies have reported that IGFBP3 promotes the proliferation of Hu sheep skeletal muscle cells and inhibits differentiation, but the downstream genes that bind to it have not been reported yet. We predicted the target genes of IGFBP3 through RNAct and sequencing data, and verified by qPCR and RIP（RNA Immunoprecipitation）experiments, and demonstrated GNAI2 （G protein subunit alpha i2）as one of the target gene of IGFBP3. After interference with siRNA, we carried out qPCR, CCK8, EdU, and immunofluorescence experiments, and found that GNAI2 can promote the proliferation and inhibit differentiation of Hu sheep skeletal muscle cells. This study revealed the effects of GNAI2 and provided one of the regulatory mechanisms of IGFBP3 protein underlying sheep muscle development.

SRY-box transcription factor 18 ( SOX18 ) is known to play a crucial role in the growth and development of hair follicles (HF) in both humans and mice. However, the specific effect of SOX18 on sheep hair follicles remains largely unknown. In our previous study, we observed that SOX18 was specifically expressed within dermal papilla cells (DPCs) in ovine hair follicles, leading us to investigate its potential role in the growth of hair follicles in sheep. In the present study, we aimed to examine the effect of SOX18 in DPCs and preliminarily study its regulatory mechanism through RNA-seq. We initially found that the overexpression of SOX18 promoted the proliferation of DPCs compared to the negative control group, while the interference of SOX18 had the opposite effect. To gain further insight into the regulatory mechanism of SOX18 , we conducted RNA-seq analysis after knocking down SOX18 in Hu sheep DPCs. The result showed that the Wnt/β-Catenin signaling pathway was involved in the growth process of DPC after SOX18 knockdown. Subsequently, we investigated the effect of SOX18 on the Wnt/β-Catenin signaling pathway in DPCs using TOP/FOP-flash, qRT-PCR, and Western blot (WB) analysis. Our data demonstrated that SOX18 could activate the Wnt/β-Catenin signaling pathway in DPCs. Additionally, we observed that SOX18 could rescue the proliferation of DPCs after inhibiting the Wnt/β-Catenin signaling pathway. These findings underscore the essential role of SOX18 as a functional molecule governing the proliferation of DPCs. Additionally, these findings also greatly enhance our understanding of the role of SOX18 in the proliferation of DPCs and the growth of wool in Hu sheep.

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.

Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG . Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR ® ) technique. As a result, a notable decrease ( p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease ( p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased ( p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly ( p < 0.05 or p < 0.01) reduced compared with the control group on days 5-7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly ( p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly ( p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits.

CUT-like homeobox 1 ( CUX1 ) has been proven to be a key regulator in sheep hair follicle development. In our previous study, CUX1 was identified as a differential expressed gene between Hu sheep lambskin with small wave patterns (SM) and straight wool patterns (ST); however, the exact molecular mechanism of CUX1 expression has been obscure. As DNA methylation can regulate the gene expression, the potential association between CUX1 core promotor region methylation and lambskin pattern in Hu sheep was explored in the present study. The results show that the core promoter region of CUX1 was present at (-1601-(-1) bp) upstream of the transcription start site. A repressive region (-1151-(-751) bp) was also detected, which had a strong inhibitory effect on CUX1 promoter activity. Bisulfite amplicon sequencing revealed that no significant difference was detected between the methylation levels of CUX1 core promoter region in SM tissues and ST tissues. Although the data demonstrated the differential expression of CUX1 between SM and ST probably has no association with DNA methylation, the identification of the core region and a potential repressive region of CUX1 promoter can enrich the role of CUX1 in Hu sheep hair follicle development.

In our previous study of Hu sheep hair follicles, we found that CRABP2 was highly expressed in DPCs, which suggested that CRABP2 may influence the number of DPCs. In the present study, we aimed to understand the effect of CRABP2 in Hu sheep dermal papilla cells (DPCs). First, we explored the influence of CRABP2 on the ability of Hu sheep DPCs' proliferation. Based on the results obtained from some experiments, such as CCK-8, EDU, qPCR, and Western blot experiment, we found that the overexpression of CRABP2 facilitated the proliferation of DPCs compared to the negative control group. Then, we also detected the effect of CRABP2 on the Wnt/β-catenin pathway based on the important function of the Wnt/β-catenin pathway in hair follicles. The results showed that CRABP2 could activate the Wnt/β-catenin pathway in DPCs, and it rescues the proliferation of DPCs when the Wnt/β-catenin pathway was inhibited. In summary, our findings indicate that CRABP2 is a vital functional gene in the proliferation of Hu sheep DPCs. Our study will be of great use for revealing the roles of CRABP2 in the hair follicles of Hu sheep.

Background: Sharply increased beef consumption is propelling the genetic improvement projects of beef cattle in China. Three-dimensional genome structure is confirmed to be an important layer of transcription regulation. Although genome-wide interaction data of several livestock species have already been produced, the genome structure states and its regulatory rules in cattle muscle are still limited., Results: Here we present the first 3D genome data in Longissimus dorsi muscle of fetal and adult cattle (Bos taurus). We showed that compartments, topologically associating domains (TADs), and loop undergo re-organization and the structure dynamics were consistent with transcriptomic divergence during muscle development. Furthermore, we annotated cis-regulatory elements in cattle genome during myogenesis and demonstrated the enrichments of promoter and enhancer in selection sweeps. We further validated the regulatory function of one HMGA2 intronic enhancer near a strong sweep region on primary bovine myoblast proliferation., Conclusions: Our data provide key insights of the regulatory function of high order chromatin structure and cattle myogenic biology, which will benefit the progress of genetic improvement of beef cattle., (© 2023. The Author(s).)

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.

Escherichia coli ( E. coli ) F17 is one of the most common pathogens causing diarrhea in farm livestock. In the previous study, we accessed the transcriptomic and microbiomic profile of E. coli F17-antagonism (AN) and -sensitive (SE) lambs; however, the biological mechanism underlying E. coli F17 infection has not been fully elucidated. Therefore, the present study first analyzed the metabolite data obtained with UHPLC-MS/MS. A total of 1957 metabolites were profiled in the present study, and 11 differential metabolites were identified between E. coli F17 AN and SE lambs (i.e., FAHFAs and propionylcarnitine). Functional enrichment analyses showed that most of the identified metabolites were related to the lipid metabolism. Then, we presented a machine-learning approach (Random Forest) to integrate the microbiome, metabolome and transcriptome data, which identified subsets of potential biomarkers for E. coli F17 infection (i.e., GlcADG 18:0-18:2, ethylmalonic acid and FBLIM1 ); furthermore, the PCCs were calculated and the interaction network was constructed to gain insight into the crosstalk between the genes, metabolites and bacteria in E. coli F17 AN/SE lambs. By combing classic statistical approaches and a machine-learning approach, our results revealed subsets of metabolites, genes and bacteria that could be potentially developed as candidate biomarkers for E. coli F17 infection in lambs.

Dermal papilla cell (DPC), one of the key cell types during hair follicle development and regeneration, specifies hair size, shape and cycling. It is also an important in vitro screening model for hair growth. Although some characteristics of DPCs, such as agglutinative growth and marker genes, have been studied in mice and humans, the intrinsic properties of ovine DPCs and the regulatory mechanism of the intrinsic properties during continued culture in vitro remained unknown. In this study, based on our previous single-cell transcriptome sequencing on sheep lambskin, we verified SOX18 and PDGFRA as the novel marker genes of ovine DPCs through immunofluorescence staining on skin sections and cultured DPCs. Using continued cell culture and alkaline phosphatase staining, we found that different from mice and humans, ovine DPCs exhibit particularly robust and stable aggregation with unbated alkaline phosphatase activity till 30 passages during continued culture in vitro . Also, we found that the expression of some marker genes and the activity of Wnt/β-catenin signaling differ between early passaged DPCs and multiple passaged DPCs. Further, using Wnt/β-catenin agonist and antagonist, we demonstrated that Wnt/β-catenin signaling could regulate cell aggregation and alkaline phosphatase activity of ovine DPCs through regulating FGF and IGF signaling. This study provides the basis for isolating ovine DPCs and defines their intrinsic properties, which contribute to improving wool performance and medicine of hair regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Hu, He, Gu, Cao, Yuan, Lv, Getachew, Quan and Sun.)

CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the DNA-binding protein homology family. Studies have shown that CUX1 is a transcription factor that plays an important role in the growth and development of hair follicles. The aim of this study was to investigate the effect of CUX1 on the proliferation of Hu sheep dermal papilla cells (DPCs) to reveal the role of CUX1 in hair follicle growth and development. First, the coding sequence (CDS) of CUX1 was amplified by PCR, and then CUX1 was overexpressed and knocked down in DPCs. A Cell Counting Kit-8 (CCK8), 5-ethynyl-2-deoxyuridine (EdU), and cell cycle assays were used to detect the changes in the proliferation and cell cycle of DPCs. Finally, the effects of overexpression and knockdown of CUX1 in DPCs on the expression of WNT10 , MMP7 , C-JUN , and other key genes in the Wnt/β-catenin signaling pathway were detected by RT-qPCR. The results showed that the 2034-bp CDS of CUX1 was successfully amplified. Overexpression of CUX1 enhanced the proliferative state of DPCs, significantly increased the number of S-phase cells, and decreased the number of G0/G1-phase cells ( p < 0.05). CUX1 knockdown had the opposite effects. It was found that the expression of MMP7 , CCND1 (both p < 0.05), PPARD , and FOSL1 (both p < 0.01) increased significantly after overexpression of CUX1 in DPCs, while the expression of CTNNB1 ( p < 0.05), C-JUN , PPARD , CCND1 , and FOSL1 (all p < 0.01) decreased significantly. In conclusion, CUX1 promotes proliferation of DPCs and affects the expression of key genes of the Wnt/β-catenin signaling pathway. The present study provides a theoretical basis to elucidate the mechanism underlying hair follicle development and lambskin curl pattern formation in Hu sheep.

Sheep milk production is a dynamic and multifactorial trait regulated by diverse biological mechanisms. To improve the quality and production of sheep milk, it is necessary to understand the underlying non-coding transcriptomic mechanisms. In this study, ribonucleic acid-sequencing (RNA-seq) was used to profile the expression of microRNAs (miRNAs) and circular RNAs (circRNAs) in the sheep mammary gland at three key lactation time points (perinatal period, PP; early lactation, EL; and peak lactation, PL). A total of 2,369 novel circRNAs and 272 miRNAs were profiled, of which 348, 373, and 36 differentially expressed (DE) circRNAs and 30, 34, and 7 DE miRNAs were detected in the comparison of EL vs. PP, PL vs. PP, and PL vs. EL, respectively. A series of bioinformatics analyses including functional enrichment, machine learning prediction, and competing endogenous RNA (ceRNA) network analyses were conducted to identify subsets of the potential candidate miRNAs (e.g., oar&#95;miR&#95;148a, oar&#95;miR&#95;362, and oar&#95;miR&#95;432) and circRNAs (e.g., novel&#95;circ&#95;0011066, novel&#95;circ&#95;0010460, and novel&#95;circ&#95;0006589) involved in sheep mammary gland development. Taken together, this study offers a window into the dynamics of non-coding transcriptomes that occur during sheep lactation and may provide further insights into miRNA and circRNA that influence sheep mammary gland development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Gu, Lv, Cao, Yuan, Wang and Sun.)

Previous genome-wide association studies (GWAS) have found that LAP3 may have the potential function to impact sheep muscle development. In order to further explore whether LAP3 expression has an important role in the development of sheep embryonic myoblasts, we conducted the spatiotemporal expression profile analysis of LAP3 at the tissue and cellular level. Then we used small interfering RNA and eukaryotic recombinant vectors to perform gain/loss-of-function analysis of LAP3 . CCK-8 detection, EdU staining, and flow cytometry were used to investigate the impact of LAP3 knockdown or overexpression on the proliferation of embryonic myoblasts. In addition, cell phenotype observation, MyHC indirect immunofluorescence, and quantitative detection of the expression changes of myogenic regulatory factors (MRFs) were used to explore the effect of LAP3 on myogenic differentiation. The results showed that the LAP3 expression level in muscle tissue of fetuses was significantly higher than that in newborn lambs and adult sheep, and its expression level on day 3 of differentiation was also significantly higher than that in the proliferation phase and other differentiation time points. LAP3 silencing could significantly increase cell viability and EdU-positive cells, as well as prolonging the length of S phase of myoblasts to promote proliferation, while the results were reversed when LAP3 was overexpressed. Moreover, LAP3 silencing significantly hindered myotube formation and down-regulated the expression levels of MRFs from day 5 to day 7 of terminal differentiation, while the results were reversed when LAP3 was highly expressed. Overall, our results suggested that the expression of LAP3 impacts on the development of sheep embryonic myoblasts which provides an important theoretical basis for molecular breeding of meat production in sheep.

Early growth response factor 1 (EGR1) is a zinc-finger transcription factor that plays a vital role in the development of hair follicles. According to our previous studies, EGR1 is a transcriptional promoter of the bone morphogenetic protein 7 (BMP7), a candidate gene involved in the proliferation of dermal papilla cells. Since hair follicles are the basis of lambskin pattern formation and dermal papilla cells (DPCs) act on hair follicle growth, in order to elucidate the role of EGR1 and hair follicles, this study aimed to investigate the biological role of EGR1 in DPCs. In our study, the EGR1 coding sequence (CDS) region was firstly cloned by polymerase chain reaction, and bioinformatics analysis was performed. Then, the function of EGR1 was detected by 5-ethynyl-2’-deoxyuridine (EDU) and Cell Counting Kit-8 (CCK8), and Western blot (WB) was conducted to analyze the cellular effect of EGR1 on DPCs. The proliferative effect of EGR1 on DPCs was also further confirmed by detecting its expression by qPCR and WB on marker genes of proliferation, including PCNA and CDK2. The sequence of the EGR1 CDS region of a lamb was successfully cloned, and its nucleic acid sequence was analyzed and found to be highly homologous to Rattus norvegicus, Mus musculus, Bos taurus and Homo sapiens. Predictive analysis of the protein encoded by EGR1 revealed that it is an extra-membrane protein, and not a secretory protein, with subcellular localization in the nucleus and cytoplasm. The proliferative effect of DPCs was significantly stronger (p < 0.01) in EGR1 up-regulated DPCs compared to the controls, while the opposite result was observed in EGR1 down-regulated DPCs. Markers of proliferation including PCNA and CDK2 also appeared to be differentially upregulated in EGR1 gene overexpression compared to the controls, with the opposite result in EGR1 gene downregulation. In summary, our study revealed that EGR1 promotes the proliferation of DPCs, and we speculate that EGR1 may be closely associated with hair follicle growth and development.

It has long been recognized that enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for vomiting and diarrhea. E. coli F17, a main subtype of ETEC, is characterized by high morbidity and mortality in young livestock. However, the transcriptomic basis underlying E. coli F17 infection has not been fully understood. In the present study, RNA sequencing was conducted to explore the expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in the jejunum of lambs who were identified as resistant or sensitive to E. coli F17 that was obtained in a challenge experiment. A total of 772 differentially expressed (DE) mRNAs and 190 DE lncRNAs were detected between the E. coli F17-resistance and E. coli F17-sensitive lambs (i.e., TFF2, LOC105606142, OLFM4, LYPD8, REG4, APOA4 , TCONS&#95;00223467, and TCONS&#95;00241897). Then, a two-step machine learning approach (RX) combination Random Forest and Extreme Gradient Boosting were performed, which identified 16 mRNAs and 17 lncRNAs as potential biomarkers, within which PPP2R3A and TCONS&#95;00182693 were prioritized as key biomarkers involved in E. coli F17 infection. Furthermore, functional enrichment analysis showed that peroxisome proliferator-activated receptor (PPAR) pathway was significantly enriched in response to E. coli F17 infection. Our finding will help to improve the knowledge of the mechanisms underlying E. coli F17 infection and may provide novel targets for future treatment of E. coli F17 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Lv, Zhang, Hu, Cao, Ren, Getachew, Mwacharo, Haile and Sun.)

Tenderness is an important indicator of meat quality. Novel isoforms associated with meat tenderness and the role of the CCCTC-binding factor (CTCF) in regulating alternative splicing to produce isoforms in sheep are largely unknown. The current project studied six sheep from two crossbred populations (Dorper × Hu × Hu, DHH and Dorper × Dorper × Hu, DDH) with divergent meat tenderness. Pooled Iso-seq data were used to annotate the sheep genomes. Then, the updated genome annotation and six RNA-seq data were combined to identify differentially expressed isoforms (DEIs) in muscles between DHH and DDH. These data were also combined with peaks detected from CTCF ChIP-seq data to investigate the regulatory role of CTCF for the alternative splicing. As a result, a total of 624 DEIs were identified between DDH and DHH. For example, isoform 7.524.18 transcribed from CAPN3 may be associated with meat tenderness. In addition, a total of 86 genes were overlapped between genes with transcribed DEIs and genes in differential peaks identified by CTCF ChIP-seq. Among these overlapped genes, ANKRD23 produces different isoforms which may be regulated by CTCF via methylation. As preliminary research, our results identified novel isoforms associated with meat tenderness and revealed the possible regulating mechanisms of alternative splicing to produce isoforms.

MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group's previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs' proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle.

It has long been recognized that enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for vomiting and diarrhea. E. coli F17, a main subtype of ETEC, is characterized by high morbidity and mortality in young livestock. However, the transcriptomic basis underlying E. coli F17 infection has not been fully understood. In this study, RNA sequencing was performed to explore the expression profiles of circRNAs and miRNAs in the jejunum of E. coli F17-antagonism (AN) and -sensitive (SE) lambs. A total of 16,534 circRNAs and 271 miRNAs (125 novel miRNAs and 146 annotated miRNAs) were screened, and 214 differentially expressed (DE) circRNAs and 53 DE miRNAs were detected between the AN and SE lambs (i.e., novel&#95;circ&#95;0025840, novel&#95;circ&#95;0022779, novel&#95;miR&#95;107, miR-10b). Functional enrichment analyses showed that source genes of DE circRNAs were mainly involved in metabolic-related pathways, while target genes of DE miRNAs were mainly enriched in the immune response pathways. Then, a two-step machine learning approach combining Random Forest (RF) and XGBoost (candidates were first selected by RF and further assessed by XGBoost) was performed, which identified 44 circRNAs and 39 miRNAs as potential biomarkers (i.e., novel&#95;circ&#95;0000180, novel&#95;circ&#95;0000365, novel&#95;miR&#95;192, oar-miR-496-3p) for E. coli infection. Furthermore, circRNA-related and lncRNA-related ceRNA networks were constructed, containing 46 circRNA-miRNA-mRNA competing triplets and 630 lncRNA-miRNA-mRNA competing triplets, respectively. By conducting a serious of bioinformatic analyses, our results revealed important circRNAs and miRNAs that could be potentially developed as candidate biomarkers for intestinal inflammatory response against E. coli F17 infection; our study can provide novel insights into the underlying mechanisms of intestinal immunity.

Hair follicle development is closely associated with wool curvature. Current studies reveal the crucial role of microRNAs (miRNAs) in hair follicle growth and development. However, few studies are known regarding their role in wool curvature. To reveal the potential roles of miRNAs in Hu sheep lambskin with different patterns, a total of 37 differentially expressed (DE) miRNAs were identified in hair follicles between small waves (SM) and straight wool (ST) groups using RNA-seq. Through functional enrichment and miRNA-mRNA co-expression analysis, some key miRNAs (oar-miR-143, oar-miR-200b, oar-miR-10a, oar-miR-181a, oar-miR-10b, oar-miR-125b, etc.) and miRNA-mRNA pairs (miR-125b target CD34 , miR-181a target FGF12 , LMO3 , miR-200b target ZNF536 , etc.) were identified. Though direct or indirect ways affecting hair follicle development, these miRNAs and mRNAs may have possible effects on wool curvature, and this study thus provides valuable insight on potential pattern formation.

Bone morphogenetic proteins (BMPs) are the structurally similar and highly conserved type of functional proteins that play an important role in hair follicle growth and development. BMP7 was a differentially expressed gene in different patterns of Hu sheep lambskin identified using Agilent microarray. Since hair follicle is the basis of pattern formation of lambskin, and its growth and development is governed by dermal papilla cells (DPCs), to clarify the role of BMP7 and hair follicle, our study was designed to investigate the regulation between BMP7 and DPCs. Firstly, the CDS region of BMP7 was cloned by 3'Race and PCR in Hu sheep and performed serious of bioinformatic analysis. Then, the effects of BMP7 on DPCs were analyzed after overexpression and interference of BMP7 in dermal papilla cells by CCK8, EdU, and PI assay. Additionally, qPCR was also conducted to clarify the relationship between BMP7 and the TGF-β/Smad signaling pathway. A total of 1296 bp of the BMP7 CDS region sequence was sucessfully cloned in Hu sheep, encoding a signal peptide of 431 amino acids, molecular weight was 49,316.9 Da and the isoelectric point (Pi) was 7.75. Nucleotide sequencing analysis of BMP7 revealed that Hu sheep had high homology with Bos taurus, Homo sapiens, and Canis lupus familiaris . Structure domain prediction showed that TGF-β superfamily domain exist between 330th-431th amino acid, BMP7 protein is a secreted protein. In BMP7 up-regulated DPCs, DPCs proliferation rate and cell cycle were significantly higher than that of NC group ( p < 0.05). Meanwhile, the expression level of Smad3 , Smad4 , Samd6 , and TGF-β1 in TGF-β/Smad signaling pathway were significantly lower than that in NC group ( p < 0.05). In BMP7 down-regulated DPCs, it presented the opposite result. In conclusion, our study showed that BMP7 had a positive effect on DPCs by accelerating the proliferation and cell cycle of DPCs, and hypothesized that regulate hair follicles growth and development via TGF-β/Smad signaling pathway. These findings may provide a synergistic target for the subsequent research of hair follicle growth and development.

The growth and development of skeletal muscle require a series of regulatory factors. MiRNA is a non-coding RNA with a length of about 22 nt, which can inhibit the expression of mRNA and plays an important role in the growth and development of muscle cells. The role of miR-22-3p in C2C12 cells and porcine skeletal muscle has been reported, but it has not been verified in Hu sheep skeletal muscle. Through qPCR, CCK-8, EdU and cell cycle studies, we found that overexpression of miR-22-3p inhibited proliferation of skeletal muscle cells ( p < 0.01). The results of qPCR and immunofluorescence showed that overexpression of miR-22-3p promoted differentiation of skeletal muscle cells ( p < 0.01), while the results of inhibiting the expression of miR-22-3p were the opposite. These results suggested that miR-22-3p functions in growth and development of sheep skeletal muscle cells. Bioinformatic analysis with mirDIP, miRTargets, and RNAhybrid software suggested IGFBP3 was the target of miR-22-3p, which was confirmed by dual-luciferase reporter system assay. IGFBP3 is highly expressed in sheep skeletal muscle cells. Overexpression of IGFBP3 was found to promote proliferation of skeletal muscle cells indicated by qPCR, CCK-8, EdU, and cell cycle studies ( p < 0.01). The results of qPCR and immunofluorescence experiments proved that overexpression of IGFBP3 inhibited differentiation of skeletal muscle cells ( p < 0.01), while the results of interfering IGFBP3 with siRNA were the opposite. These results indicate that miR-22-3p is involved in proliferation and differentiation of skeletal muscle cells by targeting IGFBP3 .

Escherichia coli ( E. coli ) F17 is a member of enterotoxigenic Escherichia coli , which can cause massive diarrhea and high mortality in newborn lambs. β-defensin is mainly produced by the epithelial tissue of the gastrointestinal tract in response to microbial infection. However, the molecular mechanism of sheep β-defensin 2 ( SBD -2) against E. coli F17 remains unclear. This study aims to reveal the antibacterial ability of SBD -2 against E. coli F17 infection in sheep. Firstly, we established the culture system of ovine intestinal epithelial cells (OIECs) in vitro, treated with different concentrations of E. coli F17 for an indicated time. Secondly, we performed RNA interference and overexpression to investigate the effect of SBD -2 expression on E. coli F17 adhesion to OIECs. Finally, inhibitors of NF-κB and MAPK pathways were pre-treated to explore the possible relationship involving in E. coli F17 infection regulating SBD -2 expression. The results showed that E. coli F17 markedly ( p < 0.01) upregulated the expression levels of SBD-2 mRNA and protein in a concentration- and time-dependent manner. Overexpression of SBD -2 contributed to enhancing E. coli F17 resistance in OIECs, while silencing SBD -2 dramatically improved the adhesion of E. coli F17 to OIECs ( p < 0.05 or p < 0.01). Furthermore, E. coli F17 stimulated SBD -2 expression was obviously decreased by pre-treatment with NF-κB inhibitor PDTC, p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 ( p < 0.05 or p < 0.01). Interestingly, adhesion of E. coli F17 to OIECs were highly enhanced by pre-treated with PDTC, SB202190 and PD98095. Our data suggested that SBD -2 could inhibit E. coli F17 infection in OIECs, possibly through NF-κB and MAPK signaling pathways. Our results provide useful theoretical basis on developing anti-infective drug and breeding for E. coli diarrhea disease-resistant sheep.

Annotating regulatory elements could benefit the interpretation of the molecular mechanism of genome-wide association study (GWAS) hits. In this work, we performed transposase-accessible chromatin with sequencing (ATAC-seq) to annotate the cattle muscle genome's functional elements. A total of 10,023 and 11,360 peaks were revealed in muscle genomes of adult and embryo cattle, respectively. The two peak sets produced 8,850 differentially accessible regions (DARs), including 2,515 promoters and 4,319 putative enhancers. These functional elements were associated with the cell cycle, muscle development, and lipid metabolism. A total of 15 putative enhancers were selected for a dual-luciferase reporter assay, and 12 of them showed enhancer activity in cattle myoblasts. Interestingly, the GeneHancer database has annotated the interactions of eight active enhancers with gene promoters, such as embryo-specific peak1053 (log2FC = 1.81, embryo/adult, E/A) with ligand-dependent nuclear receptor corepressor-like protein ( LCORL ) and embryo-specific peak4218 (log2FC = 1.81) with FERM domain-containing 8 ( FRMD8 ). A total of 295 GWAS loci from the animal QTL database were mapped to 183 putative enhancers, including rs109554838 (associated with cattle body weight and average daily gain) to peak1053 and rs110294629 (associated with beef shear force and tenderness score) to peak4218. Notably, peak4218 has been found to be involved in mouse embryo development. Deleting peak4218 clearly reduced luciferase activity ( P = 3.30E-04). Our comparative enhancer map is expected to benefit the area of beef cattle breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cao, Cheng, Huang, Lan, Lei and Chen.)

Background: Nowadays, both customers and producers prefer thin-tailed fat sheep. To effectively breed for this phenotype, it is important to identify candidate genes and uncover the genetic mechanism related to tail fat deposition in sheep. Accumulating evidence suggesting that post-transcriptional modification events of precursor-messenger RNA (pre-mRNA), including alternative splicing (AS) and alternative polyadenylation (APA), may regulate tail fat deposition in sheep. Differentially expressed transcripts (DETs) analysis is a way to identify candidate genes related to tail fat deposition. However, due to the technological limitation, post-transcriptional modification events in the tail fat of sheep and DETs between thin-tailed and fat-tailed sheep remains unclear., Methods: In the present study, we applied pooled PacBio isoform sequencing (Iso-Seq) to generate transcriptomic data of tail fat tissue from six sheep (three thin-tailed sheep and three fat-tailed sheep). By comparing with reference genome, potential gene loci and novel transcripts were identified. Post-transcriptional modification events, including AS and APA, and lncRNA in sheep tail fat were uncovered using pooled Iso-Seq data. Combining Iso-Seq data with six RNA-sequencing (RNA-Seq) data, DETs between thin- and fat-tailed sheep were identified. Protein protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were implemented to investigate the potential functions of DETs., Results: In the present study, we revealed the transcriptomic complexity of the tail fat of sheep, result in 9,001 potential novel gene loci, 17,834 AS events, 5,791 APA events, and 3,764 lncRNAs. Combining Iso-Seq data with RNA-Seq data, we identified hundreds of DETs between thin- and fat-tailed sheep. Among them, 21 differentially expressed lncRNAs, such as ENSOART00020036299, ENSOART00020033641, ENSOART00020024562, ENSOART00020003848 and 9.53.1 may regulate tail fat deposition. Many novel transcripts were identified as DETs, including 15.527.13 ( DGAT2 ), 13.624.23 ( ACSS2 ), 11.689.28 ( ACLY ), 11.689.18 ( ACLY ), 11.689.14 ( ACLY ), 11.660.12 ( ACLY ), 22.289.6 ( SCD ), 22.289.3 ( SCD ) and 22.289.14 ( SCD ). Most of the identified DETs have been enriched in GO and KEGG pathways related to extracellular matrix (ECM). Our result revealed the transcriptome complexity and identified many candidate transcripts in tail fat, which could enhance the understanding of molecular mechanisms behind tail fat deposition., Competing Interests: The authors declare that they have no competing interests., (© 2021 Yuan et al.)

Copy number variation (CNV) is the most prevalent type of genetic structural variation that has been recognized as an important source of phenotypic variation in humans, animals and plants. However, the mechanisms underlying the evolution of CNVs and their function in natural or artificial selection remain unknown. Here, we generated CNV region (CNVR) datasets which were diverged or shared among cattle, goat, and sheep, including 886 individuals from 171 diverse populations. Using 9 environmental factors for genome-wide association study (GWAS), we identified a series of candidate CNVRs, including genes relating to immunity, tick resistance, multi-drug resistance, and muscle development. The number of CNVRs shared between species is significantly higher than expected (P<0.00001), and these CNVRs may be more persist than the single nucleotide polymorphisms (SNPs) shared between species. We also identified genomic regions under long-term balancing selection and uncovered the potential diversity of the selected CNVRs close to the important functional genes. This study provides the evidence that balancing selection might be more common in mammals than previously considered, and might play an important role in the daily activities of these ruminant species., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Extrachromosomal circular DNA (eccDNA), existing as double-stranded circular DNA, is derived and free from chromosomes. It is common in eukaryotes but has a strong heterogeneity in count, length, and origin. It has been demonstrated that eccDNA could function in telomere and rDNA maintenance, aging, drug resistance, tumorigenesis, and phenotypic variations of plants and animals. Here we review the current knowledge about eccDNA in category, biogenesis, recognition, and functions. We also provide perspectives on the potential implications of eccDNA in life science., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cao, Wang, Ge, Zhang, Huang and Sun.)

Copy number variation (CNV) as an important source of genetic phenotypic and variation is related to complex phenotypic traits. The aim of this study was to investigate the potential associations of BAG4 (Bcl-2-associated athanogene 4) copy numbers variations with sheep growth traits in three Chinese sheep breeds (CKS, STHS, and HS). BAG4 is located within the stature and udder attachment quantitative trait loci (QTL) in sheep. Expression profiling revealed that the BAG4 gene was widely expressed in the tissues of sheep. The distribution of BAG4 gene copy number showed that the loss of copy number was more dominant in CKS and HS which was different from that in STHS. Statistical analysis revealed that the BAG4 CNV was significantly associated with body height in CKS ( p  < 0.05), with body slanting length in HS ( p  < 0.05), and with body height and hip cross height in STHS ( p  < 0.05). The χ 2 values showed significant differences in the BAG4 CNV distribution frequency between varieties. In conclusion, the results establish the association between BAG4 CNV and sheep traits and suggest that BAG4 CNV may be a promising marker for the molecular breeding of Chinese sheep.

Copy number variations (CNVs) are an important source of genetic variation, which can affect a wide range of economic traits by diverse mechanisms. KMT2D (Lysine methyltransferase 2D) is an important positional candidate for growth traits. Quantitative trait loci (QTLs) with large effects on economically important traits cover the KMT2D gene. The KMT2D gene overlays a CNV within its exons, hence it was chosen as a crucial candidate gene to study the association between CNV and growth traits. Further, KMT2D, a major mammalian histone H3K4 mono-methyltransferase, plays a critical role in regulating development, differentiation, metabolism, and tumor suppression. Therefore, we proposed the hypothesis that KMT2D CNV may have phenotypic effects on sheep growth traits. In our study, KMT2D CNVs in three Chinese sheep breeds were detected by quantitative polymerase chain reaction (qPCR), and the loss copy was found to be the dominant genotype. Association analysis between growth traits and KMT2D CNV was also performed, which revealed that individuals with the median copy showed better performance than those with the loss copy in all three breeds. This research suggested that KMT2D CNV can be used as a promising marker for sheep molecular breeding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Chaka sheep, named after Chaka Salt Lake, are adapted to a harsh, highly saline environment. They are known for their high-grade meat quality and are a valuable genetic resource in China. Furthermore, the Chaka sheep breed has been designated a geographical symbol of agricultural products by the Chinese Ministry of Agriculture., Results: The genomes of 10 Chaka sheep were sequenced using next-generation sequencing, and compared to that of additional Chinese sheep breeds (Mongolian: Bayinbuluke and Tan; Tibetan: Oula sheep) to explore its population structure, genetic diversity and positive selection signatures. Principle component analysis and a neighbor-joining tree indicated that Chaka sheep significantly diverged from Bayinbuluke, Tan, and Oula sheep. Moreover, they were found to have descended from unique ancestors (K = 2 and K = 3) according to the structure analysis. The Chaka sheep genome demonstrated comparable genetic diversity from the other three breeds, as indicated by observed heterozygosity (Ho), expected heterozygosity (He), runs of homozygosity (ROH), linkage disequilibrium (LD) decay. The enrichment analysis revealed that in contrast to Mongolian or Tibetan lineage groups, the genes annotated by specific missense mutations of Chaka sheep were enriched with muscle structure development (GO:0061061) factors including insulin-like growth factor 1 (IGF1), growth differentiation factor 3 (GDF3), histone deacetylase 9 (HDAC9), transforming growth factor beta receptor 2 (TGFBR2), and calpain 3 (CAPN3), among others. A genome-wide scan using Fst and XP-CLR revealed a list of muscle-related genes, including neurofibromin 1 (NF1) and myomesin 1 (MYOM1), under potential selection in Chaka sheep compared with other breeds., Conclusions: The comprehensive genome-wide characterization provided the fundamental footprints for breeding and management of the Chaka sheep and confirmed that they harbor unique genetic resources.

Copy number variations (CNVs) are important source of genetic variation, which can affect diverse economic traits through a variety of mechanisms. In addition, genome scan can identify many quantitative trait loci (QTLs) for the economic traits, while genome-wide association studies (GWAS) can localize genetic variants associated with the phenotypic variations. Here, we developed a method called GWAScore which collected GWAS summary data to identify potential candidates, and integrated CNVs into QTLs and high confidence GWAScore regions to detect crucial CNV markers for sheep growth traits. We got 197 candidate genes which were overlapping with the candidate CNVs. Some crucial genes ( MYLK3 , TTC29 , HERC6 , ABCG2 , RUNX1 , etc.) showed significantly elevated GWAScore peaks than other candidate genes. In this study, we developed the GWAScore method to excavate the potential value of candidate genes as markers for the sheep molecular breeding., (Copyright © 2020 Cheng, Cao, Hanif, Pi, Hu, Huang, Lan, Lei and Chen.)

Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Recently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples and constructed a circRNA-miRNA-mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and small interfering RNAs (siRNAs) that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, western blotting, and qRT-PCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual-luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and they also expand our understanding of circRNA functions in mammals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway., (Copyright © 2019. Published by Elsevier Inc.)

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs that are generated by reverse-splicing of precursor mRNA derived from various genes in mammals. Despite low expression, recent studies have shown that circRNA plays an important role in skeletal muscle myogenesis with competing endogenous RNA (ceRNA) functions. However, the potential regulatory role of circRNAs and interactions with miRNAs remain largely unexplored, and the function of circRNAs as miRNA sponges is not yet generally accepted. In this review, we outline the biogenesis and ceRNA mechanisms of circRNAs as well as their involvement in skeletal muscle myogenesis and discuss the conflicting conclusions of recent circRNA-ceRNA studies., (Copyright © 2019. Published by Elsevier Ltd.)

Copy number variations (CNVs) are gains and losses of genomic sequence of more than 50 bp between two individuals of a species. Also, CNV is considered to be one of the main elements affecting the phenotypic diversity and evolutionary adaptation of animals. ORMDL sphingolipid biosynthesis regulator 1 ( ORMDL1 ) is a protein-coding gene associated with diseases and development. In our study, the polymorphism of ORMDL1 gene copy numbers in four Chinese sheep breeds (abbreviated CK, HU, STH, and LTH) was detected. In addition, we analyzed the transcriptional expression level of ORMDL1 gene in different tissues of sheep and examined the association of ORMDL1 CNV with growth traits. The statistical analysis revealed that ORMDL1 CNV was remarkably correlated with body height, heart girth, and circumference of cannon bone in HU sheep ( P < 0.05 ), and there are significant effects on body weight, body height, body length, chest depth, and height of hip cross in STH sheep ( P < 0.05 ). In conclusion, our results provide a basis for the relationship between CNV of ORMDL1 gene and sheep growth traits, suggesting that ORMDL1 CNV may be considered a promising marker for the molecular breeding of Chinese sheep., (Copyright: © 2019 Xiaogang Wang et al.)

Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA, lncFAM200B , was differentially expressed in embryonic, neonatal, and adult cattle skeletal muscles. The main aim of this study was to investigate the molecular and expression characteristics of lncFAM200B along with its crucial genetic variations. Our results showed that bovine lncFAM200B was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that lncFAM200B promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that lncFAM200B was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the lncFAM200B expression trend positively correlated with that of MyoG and Myf5 in myoblast proliferation and differential stages. To identify the promoter active region of lncFAM200B , we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing lncFAM200B promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that lncFAM200B promoter active region was from -403 to -139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that lncFAM200B is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle., (Copyright © 2019 Zhang, Kang, Sun, Cao, Pan, Dang, Lei, Chen and Lan.)

Development of flexible surface-enhanced Raman spectroscopy (SERS) substrate with controllable "hot spots" has spurred increasing interest because of its unique structure and plasmonic properties. Here, charged poly(vinyl alcohol) microgels containing silver nanoparticles are developed by using microfluidic emulsification to produce a "smart" SERS sensor with charge screening and signals amplification. Importantly, this charged microgel enables the selective concentration of counter-charged molecules and induces the formation of assembled arrays at an immiscible liquid-liquid interface because of the electrostatic interaction. The SERS-active microgels arrays possess controllable structures and facilitate on-site determination of charged pesticides with an enhancement factor of 5.0 × 10 5 . Such nanostructures present the ease of assembly, stability, and reproducibility which allow multiplex detection of analytes at aqueous and organic phases without any pretreatment of the complex matrix samples. The interfacial sensing platform for on-site SERS analysis of charged pesticides will open vast possibilities for a wide range of in-field applications.

Myogenesis is a complex and precisely orchestrated process that is highly regulated by several non-coding RNAs and signal pathways. Circular RNAs (circRNAs) represent a novel subclass of endogenous non-coding RNAs that have been identified in multiple species and tissues and play a vital role in post-transcriptional regulation in eukaryotes, but the precise molecular mechanism of action remains largely unknown. Here, we screened a candidate circRNA derived from the SNX29 gene, termed circSNX29 from our previous circRNAs sequencing data of bovine skeletal muscle, and further characterized its regulation and function during muscle development. The overexpression of circSNX29 facilitated myoblasts differentiation and inhibited cell proliferation. Computational analysis using RNAhybrid showed the potential for circSNX29 to sponge to miR-744 with nine potential binding sites. We tested this via a luciferase screening assay and found that circSNX29 directly interacted with miR-744 and downregulation of miR-744 efficiently reversed the suppression of Wnt5a and CaMKIIδ. Importantly, through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, Fluo-4, AM, cell permeant-calcium ion fluorescent probing, and western blotting assays, we found that overexpression of Wnt5a and circSNX29 activated the non-canonical Wnt5a/Ca 2+ pathway. Overall, the evidence generated by our study elucidates the regulatory mechanisms of circSNX29 to function as a sponge for miRNA-744 in bovine primary myoblasts., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The potassium inwardly rectifying channel, subfamily J, member 12 ( KCNJ12 ) gene is a promising candidate for economic traits because of its crucial roles in myoblast development. Here, a missense mutation (Cys > Arg) was first detected to be located in exon 3 of KCNJ12 from three Chinese cattle breeds by DNA-pool sequencing. Then, we performed an association analysis of this single-nucleotide polymorphism (SNP) with stature in three Chinese cattle populations ( n = 820). A significantly positive correlation was revealed by a reduced animal general linear model and the CC genotype was the most favorable in three breeds. Further, we measured the expression profile of the KCNJ12 gene in various cattle tissues and primary bovine skeletal muscle cells. Ubiquitous expression with high abundance in muscle was observed. Further, in primary bovine skeletal muscle cells, the KCNJ12 mRNA expression was gradually up-regulated in differentiation medium (DM) compared with that in growth medium (GM), suggesting that the KCNJ12 gene is involved in bovine myocyte differentiation. Conclusively, the KCNJ12 gene is a functional candidate gene which can be used as a molecular marker for cattle breeding.

Skeletal muscle is the most abundant tissue in the body. The development of skeletal muscle cell is complex and affected by many factors. A sea of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. MiR-208b, a muscle-specific miRNA, was reported to have a connection with fiber type determination. However, whether miR-208b has effect on proliferation of muscle cell was under ascertained. In our study, cyclin-dependent kinase inhibitor 1A (CDKN1A), which participates in cell cycle regulation, was predicted and then validated as one target gene of miR-208b. We found that overexpression of miR-208b increased the expression of cyclin D1, cyclin E1, and cyclin-dependent kinase 2 at the levels of messenger RNA and protein in cattle primary myoblasts in vivo and in vitro. Flow cytometry showed that forced expression of miR-208b increased the percentage of cells at the S phase and decreased the percentage of cells at the G0/G1 phase. These results indicated that miR-208b participates in the cell cycle regulation of cattle primary myoblast cells. 5-Ethynyl-20-deoxyuridine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that overexpression of miR-208b promoted the proliferation of cattle primary myoblasts. Therefore, we conclude that miR-208b participates in the cell cycle and proliferation regulation of cattle primary skeletal muscle cell through the posttranscriptional downregulation of CDKN1A., (© 2018 Wiley Periodicals, Inc.)

The detection method based on the mathematical expectation (ME) strategy is fast and accuracy for low frequency mutation screening in large samples. Previous studies have found that the 14-bp insertion/deletion (indel) variants of the 3' untranslated region (3' UTR) within bovine PRNP gene have been characterized with low frequency (≤5%) in global breeds outside China, which has not been determined in Chinese cattle breeds yet. Therefore, this study aimed to identify the 14-bp indel within PRNP gene in 5 major Chinese indigenous cattle breeds and to evaluate its associations with phenotypic traits. It was the first time to use ME strategy to detect low frequency indel polymorphisms and found that minor allele frequency was 0.038 (Qinchuan), 0.033 (Xianan), 0.013 (Nanyang), 0.003 (Jiaxian), and zero (Ji'an), respectively. Compared to the traditional detection method by which the sample was screened one by one, the reaction time by using the ME method was decreased 62.5%, 64.9%, 77.6%, 88.9% and 66.4%, respectively. In addition, the 14-bp indel was significantly associated with the growth traits in 2 cattle breeds, with the body length of Qinchuan cattle as well as the body weight and waistline of Xianan cattle. Our results have uncovered that the method based on ME strategy is rapid, reliable, and cost-effective for detecting the low frequency mutation as well as our findings provide a potential valuable theoretical basis for the marker-assisted selection (MAS) in beef cattle.

NPC1 gene is an important gene closely related to the Niemann-Pick type C (NPC). Mutations in the NPC1 gene tend to cause Niemann-Pick type C, a lysosomal storage disorder. Previous studies have shown that NPC1 protein plays an important role in subcellular lipid transport, homeostasis, platelet function and formation, which are basic metabolic activities in the process of development. In this study, to explore the association between the NPC1 gene variation and body size traits in Qinchuan cattle, we detected four novel coding single nucleotide polymorphisms (cSNPs) in the bovine NPC1 gene, including one missense mutation (SNP1) and three synonymous mutations (SNP2, SNP3 and SNP4). Population genetic analyses of 518 individuals and association correlations between cSNPs and bovine body size traits were conducted in this research. A missense mutation at SNP1 locus was found to be significantly related to the heart girth, hip width and body weight (P<0.01 or P<0.05, 3.5-year-old). Two synonymous mutations at SNP2 and SNP3 loci also showed significant effects on hip width (P<0.05, 3.5-year-old). One synonymous mutation at SNP4 locus showed significant effect on body weight (P<0.05, 2.0-year-old). Combined haplotypes H2H6 and H6H6 showed significant effects on body size traits such as heart girth, hip width, and body weight (3.5-year-old, P<0.01 or P<0.05). This study provides evidence that the NPC1 gene might be involved in the regulation of bovine growth and body development, and may be considered as a candidate gene for marker assisted selection (MAS) in beef cattle breeding industry., (Copyright © 2014. Published by Elsevier B.V.)

Cell death-inducing DFFA-like effector c (CIDEC) protein, also known as fat specific protein 27 (Fsp27), is localized to lipid droplets. CIDEC protein is required for unilocular lipid droplet formation and optimal energy storage in addition to controlling lipid metabolism in adipocytes and hepatocytes. Research found that Ad-36 could induce lipid droplets in the cultured skeletal muscle cells and this process may be mediated by promoting CIDEC expression. The content of intermuscular fat is an important index for evaluation of beef quality, so the CIDEC gene appeared to be a candidate gene for regulation of intermuscular fat, however similar research for the bovine CIDEC gene is lacking. This paper examined the tissue expression profile of CIDEC gene in cattle using real-time RT-PCR to suggest that bovine CIDEC is highly expressed in adipose tissue. In addition, the Bovine CIDEC gene was cloned and inserted into the eukaryotic expression vector pET-28a(+), whereupon recombinant bovine CIDEC protein was induced and identified by Western-blot. A phylogenetic analysis showed that the animo acid sequence of bovine CIDEC was closer to mammalian CIDEC than rasorial CIDEC. We found ten single nucleotide polymorphisms sites (SNPs) in bovine CIDEC gene, of which SNP 2, 3, 4, 6 and 9, and SNP 8 and 10 were in complete linkage disequilibrium, respectively. SNP 1, 2 and 10 were used in further haplotype studies. Eight different haplotypes were identified in 973 cattle, of which haplotype 8 predominated with frequencies ranging from 42.90 to 54.30 %. This research provides a basis for future functional studies of CIDEC in cattle.