Methylation Levels in the Promoter Region of FHIT and PIAS1 Genes Associated with Mastitis Resistance in Xinjiang Brown Cattle.

Mastitis causes serious economic losses in the dairy industry, but there are no effective treatments or preventive measures. In this study, the ZRANB3, PIAS1, ACTR3, LPCAT2, MGAT5, and SLC37A2 genes in Xinjiang brown cattle, which are associated with mastitis resistance, were identified using a GWAS. Pyrosequencing analysis showed that the promoter methylation levels of the FHIT and PIAS1 genes in the mastitis group were higher and lower, respectively, than those in the healthy group (65.97 ± 19.82% and 58.00 ± 23.52%). However, the methylation level of the PIAS1 gene promoter region in the mastitis group was lower than that in the healthy group (11.48 ± 4.12% and 12.17 ± 4.25%). Meanwhile, the methylation levels of CpG3, CpG5, CpG8, and CpG15 in the promoter region of the FHIT and PIAS1 genes in the mastitis group were significantly higher than those in the healthy group (p < 0.01), respectively. RT-qPCR showed that the expression levels of the FHIT and PIAS1 genes were significantly higher in the healthy group than those in the mastitis group (p < 0.01). Correlation analysis showed that the promoter methylation level of the FHIT gene was negatively correlated with its expression. Hence, increased methylation in the promoter of the FHIT gene reduces the mastitis resistance in Xinjiang brown cattle. Finally, this study provides a reference for the molecular-marker-assisted selection of mastitis resistance in dairy cattle.

Transcriptome analysis reveals genes associated with wool fineness in merinos.

Hair/wool usually plays an important role in maintaining animal physiological activities, and the economic value of wool cannot be ignored. At present, people set higher demands on wool fineness. Hence, improving wool fineness is the concern of fine wool sheep breeding. Using RNA-Seq to screen the potential candidate genes that associate with wool fineness can provide theoretical references for fine-wool sheep breeding, and also provide us new ideas for further understand the molecular regulation mechanism of hair growth. In this study, we compared the expression pattern difference of genome-wide genes between the skin transcriptomes of Subo and Chinese Merinos. The results showed that, 16 candidate differentially expressed genes (DEGs) (Included: CACNA1S, GP5, LOC101102392, HSF5, SLITRK2, LOC101104661, CREB3L4, COL1A1, PTPRR, SFRP4, LOC443220, COL6A6, COL6A5, LAMA1, LOC114115342 and LOC101116863 genes) that may associate with wool fineness were screened, and these genes were located in signaling pathways that regulate hair follicle development, cycle or hair growth. It is worth noting that, among the 16 DEGs, COL1A1 gene has the highest expression level in Merino skins, and the fold change of LOC101116863 gene is the highest, and the structures of these two genes are both highly conserved in different species. In conclusion, we speculate that these two genes may play a key role in regulating wool fineness and respectively have similar and conserved functions in different species.

Integrated analysis of miRNAs and mRNA profiling reveals the potential roles of miRNAs in sheep hair follicle development.

BACKGROUND: Merino sheep exhibit high wool production and excellent wool quality. The fleece of Merino sheep is predominantly composed of wool fibers grown from hair follicles (HFs). The HF is a complex biological system involved in a dynamic process governed by gene regulation, and gene expression is regulated by microRNAs (miRNAs). miRNA inhibits posttranscriptional gene expression by specifically binding to target messenger RNA (mRNA) and plays an important role in regulating gene expression, the cell cycle and biological development sequences. The purpose of this study was to examine mRNA and miRNA binding to identify key miRNAs and target genes related to HF development. This will provide new and important insights into fundamental mechanisms that regulate cellular activity and cell fate decisions within and outside of the skin. RESULTS: We analyzed miRNA data in skin tissues collected from 18 Merino sheep on four embryonic days (E65, E85, E105 and E135) and two postnatal days (D7 and D30) and identified 87 differentially expressed miRNAs (DE-miRNAs). These six stages were further divided into two longer developmental stages based on heatmap cluster analysis, and the results showed that DE-mRNAs in Stage A were closely related to HF morphogenesis. A coanalysis of Stage A DE-mRNAs and DE-miRNAs revealed that 9 DE-miRNAs and 17 DE-mRNAs presented targeting relationships in Stage A. We found that miR-23b and miR-133 could target and regulate ACVR1B and WNT10A. In dermal fibroblasts, the overexpression of miR-133 significantly reduced the mRNA and protein expression levels of ACVR1B. The overexpression of miR-23b significantly reduced the mRNA and protein expression levels of WNT10A. CONCLUSION: This study provides a new reference for understanding the molecular basis of HF development and lays a foundation for further improving sheep HF breeding. miRNAs and target genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine-wool sheep.

Development and application of arachnomelia syndrome genetic detection in four Chinese dual-purpose cattle populations.

Arachnomelia syndrome (AS) is an autosomal recessive hereditary disorder in cattle, and affected calves are usually stillborn and characterized by complex anomalies. Therefore, identification of the carrier animals based on genetic tests is important for the control and elimination of this defect. The aim of this study was to build an effective workflow to routinely screen the AS mutations in bovine MOSC1 and SUOX genes and determine individuals carrying the AS mutations in four Chinese cattle populations. By combining the fluorescence-labeled PCR and capillary electrophoresis, we established a convenient and cost-effective workflow to detect two AS casual mutations simultaneously. Sanger sequencing was further used as a validation criterion and showed that 100% of the tests (37/37) had consistent results with genotype calls determined by our established workflow. Then, 582 bulls and 1-926 cows from Chinese dual-purpose cattle populations of Simmental, Sanhe, Shuxuan, and Xinjiang Brown were subjected to AS detection. The results showed that four bulls and 11 cows in the Simmental population, and six bulls and six cows in the Sanhe population were identified as AS carriers with the MOCS1 mutation c.1224_1225delCA. However, no animal was found to carry the c.363_364insG mutation in the SUOX gene. The frequencies of AS carriers were 1.08% and 1.65% in the Simmental and Sanhe populations, respectively, with a frequency of 1.076% in four populations. The pedigree analysis found that all carriers could be traced back to a common ancestor, the German Simmental sire ROMEL. Those findings suggested that this genetic defect spread into China mainly through the wide use of ROMEL. In conclusion, the occurrence of AS has not had a wide impact on the Chinese cattle industry; however, a screening system and mating strategy should be employed to gradually eliminate this recessive gene from the Chinese dual-purpose cattle population.

Gene network analysis reveals candidate genes related with the hair follicle development in sheep.

BACKGROUND: Merino sheep are the most famous fine wool sheep in the world. They have high wool production and excellent wool quality and have attracted worldwide attention. The fleece of the Merino sheep is composed predominantly of wool fibers grown from secondary wool follicles. Therefore, it is necessary to study the development of hair follicles to understand the mechanism of wool production. The hair follicle is a complex biological system involved in a dynamic process governed by gene regulation. The hair follicle development process is very complex and poorly understood. The purpose of our research is to identify candidate genes related to hair follicle development, provide a theoretical molecular breeding basis for the cultivation of fine wool sheep, and provide a reference for the problems of hair loss and alopecia areata that affect human beings. RESULTS: We analyzed mRNAs data in skin tissues of 18 Merino sheep at four embryonic days (E65, E85, E105 and E135) and two postnatal days (P7 and P30). G1 to G6 represent hair follicles developmental at six stages (i.e. E65 to P30). We identified 7879 differentially expressed genes (DEGs) and 12623 novel DEGs, revealed different expression patterns of these DEGs at six stages of hair follicle development, and demonstrated their complex interactions. DEGs with stage-specific expression were significantly enriched in epidermal differentiation and development, hair follicle development and hair follicle morphogenesis and were enriched in many pathways related to hair follicle development. The key genes (LAMA5, WNT10A, KRT25, SOSTDC1, ZDHHC21, FZD1, BMP7, LRP4, TGFß2, TMEM79, SOX10, ITGB4, KRT14, ITGA6, and GLI2) affecting hair follicle morphogenesis were identified by network analysis. CONCLUSION: This study provides a new reference for the molecular basis of hair follicle development and lays a foundation for further improving sheep hair follicle breeding. Candidate genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine wool sheep. These results are a valuable resource for biological investigations of fleece evolution in animals.

Differential Methylation and Transcriptome Integration Analysis Identified Differential Methylation Annotation Genes and Functional Research Related to Hair Follicle Development in Sheep.

Hair follicle growth and development are a complex and long-term physiological process, which is regulated by a variety of physical factors and signal pathways. Increasing the understanding of the epigenetic regulation and function of candidate genes related to hair follicle development will help to better understand the molecular regulatory mechanisms of hair follicle development. In this study, the methylated DNA immunoprecipitation sequencing (MeDIP-seq) was used to obtain the genome-wide methylation map of the hair follicular development of Super Merino sheep in six stages (fetal skin tissue at 65d, 85d, 105d, 135d, 7d, and 30d after birth). Combined with the results of previous RNA-sequencing, 65 genes were screened out that were both differential methylation and differential expression, including EDN1, LAMC2, NR1D1, RORB, MyOZ3, and WNT2 gene. Differential methylation genes were enriched in Wnt, TNF, TGF-beta, and other signaling pathways related to hair follicle development. The bisulfite sequencing PCR results and MeDIP-seq were basically consistent, indicating that the sequencing results were accurate. As a key gene in the Wnt signaling pathway, both differential methylation and expression gene identified by MeDIP-seq and RNA-seq, further exploration of the function of WNT2 gene revealed that the DNA methylation of exon 5 (CpG11 site) promoted the expression of WNT2 gene. The overexpression vector of lentivirus pLEX-MCS-WNT2 was constructed, and WNT2 gene effectively promoted the proliferation of sheep skin fibroblasts. The results showed that WNT2 gene could promote the growth and development of skin and hair follicles. The results of this study will provide a theoretical basis for further research on sheep hair follicle development and gene regulation mechanisms.

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

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

Integration of a single-step genome-wide association study with a multi-tissue transcriptome analysis provides novel insights into the genetic basis of wool and weight traits in sheep.

BACKGROUND: Genetic improvement of wool and growth traits is a major goal in the sheep industry, but their underlying genetic architecture remains elusive. To improve our understanding of these mechanisms, we conducted a weighted single-step genome-wide association study (WssGWAS) and then integrated the results with large-scale transcriptome data for five wool traits and one growth trait in Merino sheep: mean fibre diameter (MFD), coefficient of variation of the fibre diameter (CVFD), crimp number (CN), mean staple length (MSL), greasy fleece weight (GFW), and live weight (LW). RESULTS: Our dataset comprised 7135 individuals with phenotype data, among which 1217 had high-density (HD) genotype data (n = 372,534). The genotypes of 707 of these animals were imputed from the Illumina Ovine single nucleotide polymorphism (SNP) 54 BeadChip to the HD Array. The heritability of these traits ranged from 0.05 (CVFD) to 0.36 (MFD), and between-trait genetic correlations ranged from - 0.44 (CN vs. LW) to 0.77 (GFW vs. LW). By integrating the GWAS signals with RNA-seq data from 500 samples (representing 87 tissue types from 16 animals), we detected tissues that were relevant to each of the six traits, e.g. liver, muscle and the gastrointestinal (GI) tract were the most relevant tissues for LW, and leukocytes and macrophages were the most relevant cells for CN. For the six traits, 54 quantitative trait loci (QTL) were identified covering 81 candidate genes on 21 ovine autosomes. Multiple candidate genes showed strong tissue-specific expression, e.g. BNC1 (associated with MFD) and CHRNB1 (LW) were specifically expressed in skin and muscle, respectively. By conducting phenome-wide association studies (PheWAS) in humans, we found that orthologues of several of these candidate genes were significantly (FDR < 0.05) associated with similar traits in humans, e.g. BNC1 was significantly associated with MFD in sheep and with hair colour in humans, and CHRNB1 was significantly associated with LW in sheep and with body mass index in humans. CONCLUSIONS: Our findings provide novel insights into the biological and genetic mechanisms underlying wool and growth traits, and thus will contribute to the genetic improvement and gene mapping of complex traits in sheep.

The Effect of Integrating Genomic Information into Genetic Evaluations of Chinese Merino Sheep.

Genomic evaluations are a method for improving the accuracy of breeding value estimation. This study aimed to compare estimates of genetic parameters and the accuracy of breeding values for wool traits in Merino sheep between pedigree-based best linear unbiased prediction (PBLUP) and single-step genomic best linear unbiased prediction (ssGBLUP) using Bayesian inference. Data were collected from 28,391 yearlings of Chinese Merino sheep (classified in 1992-2018) at the Xinjiang Gonaisi Fine Wool Sheep-Breeding Farm, China. Subjectively-assessed wool traits, namely, spinning count (SC), crimp definition (CRIM), oil (OIL), and body size (BS), and objectively-measured traits, namely, fleece length (FL), greasy fleece weight (GFW), mean fiber diameter (MFD), crimp number (CN), and body weight pre-shearing (BWPS), were analyzed. The estimates of heritability for wool traits were low to moderate. The largest h2 values were observed for FL (0.277) and MFD (0.290) with ssGBLUP. The heritabilities estimated for wool traits with ssGBLUP were slightly higher than those obtained with PBLUP. The accuracies of breeding values were low to moderate, ranging from 0.362 to 0.573 for the whole population and from 0.318 to 0.676 for the genotyped subpopulation. The correlation between the estimated breeding values (EBVs) and genomic EBVs (GEBVs) ranged from 0.717 to 0.862 for the whole population, and the relative increase in accuracy when comparing EBVs with GEBVs ranged from 0.372% to 7.486% for these traits. However, in the genotyped population, the rank correlation between the estimates obtained with PBLUP and ssGBLUP was reduced to 0.525 to 0.769, with increases in average accuracy of 3.016% to 11.736% for the GEBVs in relation to the EBVs. Thus, genomic information could allow us to more accurately estimate the relationships between animals and improve estimates of heritability and the accuracy of breeding values by ssGBLUP.

Genome-wide identification and characterization of long non-coding RNAs expressed during sheep fetal and postnatal hair follicle development.

Long non-coding RNAs (lncRNAs), >200 nt in length, are transcribed from mammalian genomes. They play important regulatory roles in various biological processes; However, the function and expression profile of lncRNAs involved in the development of hair follicles in the fetus, have been relatively under-explored area. To investigate the specific role of lncRNAs and mRNAs that regulate hair follicle development, we herein performed a comprehensive study on the lncRNA and mRNA expression profiles of sheep at multiple embryonic days (E65, E85, E105, and E135) and six lambs aged one week (D7) and one month (D30) using RNA-seq technology. The number of genes (471 lncRNAs and 12,812 mRNAs) differentially expressed and potential targets of differentially expressed lncRNAs were predicted. Differentially expressed lncRNAs were grouped into 10 clusters based on their expression pattern by K-means clustering. Moreover, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that some differentially expressed mRNAs, such as DKK1, DSG4, FOXE1, Hoxc13, SFRP1, SFRP2, and Wnt10A overlapped with lncRNAs targets, and enriched in important hair follicle developmental pathways, including Wnt, TNF, and MAPK signaling pathways. In addition, 9 differentially expressed lncRNAs and 4 differentially expressed mRNAs were validated using quantitative real-time PCR (qRT-PCR). This study helps enrich the Ovis lncRNA databases and provides a comprehensive lncRNA transcriptome profile of fetal and postnatal skin of sheep. Additionally, it provides a foundation for further experiments on the role of lncRNAs in the regulation of hair growth in sheep.

Association analysis of polymorphisms in six keratin genes with wool traits in sheep.

OBJECTIVE: The purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals. METHODS: To explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used. RESULTS: We report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in Ile→Thr, Glu→Asp, Gly→Ala, Ala→Ser, Se→His; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p< 0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05). CONCLUSION: Our results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

WITHDRAWN:Identification of Polymorphisms and Association of Five KAP Genes with Sheep Wool Traits.

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