Two mutations at KRT74 and EDAR synergistically drive the fine-wool production in Chinese sheep.

INTRODUCTION: Fine-wool sheep are the most common breed used by the wool industry worldwide. Fine-wool sheep have over a three-fold higher follicle density and a 50% smaller fiber diameter than coarse-wool sheep. OBJECTIVES: This study aims to clarify the underlying genetic basis for the denser and finer wool phenotype in fine-wool breeds. METHOD: Whole-genome sequences of 140 samples, Ovine HD630K SNP array data of 385 samples, including fine, semi-fine, and coarse wool sheep, as well as skin transcriptomes of nine samples were integrated for genomic selection signature analysis. RESULTS: Two loci at keratin 74 (KRT74) and ectodysplasin receptor (EDAR) were revealed. Fine-scale analysis in 250 fine/semi-fine and 198 coarse wool sheep narrowed this association to one C/A missense variant of KRT74 (OAR3:133,486,008, P = 1.02E-67) and one T/C SNP in the regulatory region upstream of EDAR (OAR3:61,927,840, P = 2.50E-43). Cellular over-expression and ovine skin section staining assays confirmed that C-KRT74 activated the KRT74 protein and specifically enlarged cell size at the Huxley's layer of the inner root sheath (P < 0.01). This structure enhancement shapes the growing hair shaft into the finer wool than the wild type. Luciferase assays validated that the C-to-T mutation upregulated EDAR mRNA expression via a newly created SOX2 binding site and potentially led to the formation of more hair placodes. CONCLUSIONS: Two functional mutations driving finer and denser wool production were characterized and offered new targets for genetic breeding during wool sheep selection. This study not only provides a theoretical basis for future selection of fine wool sheep breeds but also contributes to improving the value of wool commodities.

Genome-Wide Signal Selection Analysis Revealing Genes Potentially Related to Sheep-Milk-Production Traits.

Natural selection and domestication have shaped modern sheep populations into a vast range of phenotypically diverse breeds. Among these breeds, dairy sheep have a smaller population than meat sheep and wool sheep, and less research is performed on them, but the lactation mechanism in dairy sheep is critically important for improving animal-production methods. In this study, whole-genome sequences were generated from 10 sheep breeds, including 57 high-milk-yield sheep and 44 low-milk-yield sheep, to investigate the genetic signatures of milk production in dairy sheep, and 59,864,820 valid SNPs (Single Nucleotide Polymorphisms) were kept after quality control to perform population-genetic-structure analyses, gene-detection analyses, and gene-function-validation analyses. For the population-genetic-structure analyses, we carried out PCA (Principal Component Analysis), as well as neighbor-joining tree and structure analyses to classify different sheep populations. The sheep used in our study were well distributed in ten groups, with the high-milk-yield-group populations close to each other and the low-milk-yield-group populations showing similar classifications. To perform an exact signal-selection analysis, we used three different methods to find SNPs to perform gene-annotation analyses within the 995 common regions derived from the fixation index (FST), nucleotide diversity (Ɵπ), and heterozygosity rate (ZHp) results. In total, we found 553 genes that were located in these regions. These genes mainly participate in the protein-binding pathway and the nucleoplasm-interaction pathway, as revealed by the GO- and KEGG-function-enrichment analyses. After the gene selection and function analyses, we found that FCGR3A, CTSK, CTSS, ARNT, GHR, SLC29A4, ROR1, and TNRC18 were potentially related to sheep-milk-production traits. We chose the strongly selected genes, FCGR3A, CTSK, CTSS, and ARNT during the signal-selection analysis to perform a RT-qPCR (Reale time Quantitative Polymerase Chain Reaction) experiment to validate their expression-level relationship with milk production, and the results showed that FCGR3A has a significant negative relationship with sheep-milk production, while other three genes did not show any positive or negative relations. In this study, it was discovered and proven that the candidate gene FCGR3A potentially contributes to the milk production of dairy sheep and a basis was laid for the further study of the genetic mechanism underlying the strong milk-production traits of sheep.

Transcriptome Profiling of Developing Ovine Fat Tail Tissue Reveals an Important Role for MTFP1 in Regulation of Adipogenesis.

Fat-tail sheep exhibit a unique trait whereby substantial adipose tissue accumulates in the tail, a phenotype that is advantageous in many agroecological environments. In this study, we conducted histological assays, transcriptome analysis and functional assays to examine morphogenesis, characterize gene expression, and elucidate mechanisms that regulate fat tail development. We obtained the microstructure of tail before and after fat deposition, and demonstrated that measurable fat deposition occurred by the 80-day embryo (E80) stage, earlier than other tissues. Transcriptome profiling revealed 1,058 differentially expressed genes (DEGs) with six markedly different expression trends. GSEA enrichment and other downstream analyses showed important roles for genes and pathways involving in metabolism and that mitochondrial components were specifically overexpressed in the fat tail tissue of the 70-day embryo (E70). One hundred and eighty-three genes were further identified by leading edge gene analysis, among which, 17 genes have been reported in previous studies, including EEF1D, MTFP1, PPP1CA, PDGFD. Notably, the MTFP1 gene was highly correlated with the expression of other genes and with the highest enrichment score and gene expression change. Knockdown of MTFP1 in isolated adipose derived stem cells (ADSCs) inhibited cell proliferation and migration ability, besides, promoted the process of adipogenesis in vitro.

A study using single-locus and multi-locus genome-wide association study to identify genes associated with teat number in Hu sheep.

The multiple teats trait is common in many species of mammals and is considered related to lactation ability in swine. However, in Hu sheep, related gene research is still relatively limited. In this study, a genome-wide association study was used to identify genetic markers and genes related to the number of teats in the Hu sheep population, a native Chinese sheep breed. A single marker method and several multi-locus methods were utilized. A total of 61 SNPs were found to be related to the number of teats. Among these, 11 SNPs and one SNP were consistently detected by two and three multi-locus models respectively. Four SNPs were concordantly identified between the single marker and multi-locus methods. We also performed quantitative real-time PCR testing of these identified candidate genes, identifying three genes with significantly different expression. Our study suggested that the LHFP, DPYSL2, and TDP-43 genes may be related to the number of teats in sheep. The combination of single and multi-locus GWAS detected additional SNPs not found with only one model. Our results provide new and important insights into the genetic mechanisms of the mammalian multiparous teat phenotype. These findings may be useful for future breeding and understanding the genetics of sheep and other livestock.

Temporal expression of the KISS1/GPR54 system in goats' testes and epididymides and its spatial expression in pubertal goats.

Kisspeptin, encoded by the KISS1 gene, and its receptor GPR54 are essential in puberty onset and male fertility due to their central regulatory roles. However, the roles of KISS1/GPR54 in peripheral tissues remain unclear. This study aimed to investigate the temporal expression patterns of KISS1/GPR54 in goat testes and epididymides and its spatial expression patterns in pubertal goats. Immunohistochemical analysis revealed that kisspeptin/GPR54 were localized in Leydig, Sertoli, and germ cells of pubertal goats' testis, as well as in principal and basal cells of the epididymis. RT-PCR revealed a marked variation in the KISS1/GPR54 expressions in the testes and epididymides from the age of first week to adulthood. KISS1 and GPR54 mRNA levels in testes decreased from the age of first week to two months and then increased from two months to puberty and adulthood. The KISS1 and GPR54 mRNA levels in Leydig cells decreased from the age of one week to two months and increased from two months to puberty, and then decreased from puberty to adulthood. Only GPR54 mRNA levels in the epididymides increased from the age of one week to two months and puberty, and then decreased from puberty to adulthood. RT-PCR analysis showed the different spatial expression patterns of KISS1/GPR54 in pubertal goat tissues. The KISS1 mRNA level was high in the hypothalamus, moderate in pancreas, liver, epididymis and testis; and low in the other tissues. The GPR54 expression was high in the pancreas and testis; moderate in pituitary, hypothalamus and mesenteric lymph node; and low in the other tissues. In conclusion, the KISS1/GPR54 system possessed distinct temporal expression profiles in goats' testes and epididymides, as well as different spatial expression patterns in pubertal goat tissues, which implied the possible local role of this system in goats' testes, epididymides, and other peripheral tissues.

Potent effect of KISS1-54 DNA vaccine compared with KISS1-10 DNA vaccine in inhibiting the fertility of female rats.

BACKGROUND: Most studies on immunocastration currently focused on male animals. However, immunization of male animals does not completely inhibit sexual behavior and fertility. This study aimed to compare the immunocastration effect of KISS1 DNA vaccines encoding different lengths of kisspeptins in female rats for effective castration effects on both male and female rats. METHODS: Fifteen female rats were randomly divided into three groups. The rats in T1 group or T2 group was orally given respectively KISS1-54 or KISS1-10 DNA vaccines with fused tPA signal peptide, and the control group (Group C) was orally administered with empty vector vaccine, at a dose of 5 × 109 CFU/rat at weeks 0, 3 and 6 of the study. Blood samples were collected by retroorbital bleeding before primary immunization and at weeks 3 and 9 after primary immunization. RESULTS: Both KISS1-54 and KISS1-10 DNA vaccines induced the body's humoral immune response, and the anti-kisspeptin antibody titres in the T1 group were significantly higher than that in T2 and C groups (p < 0.05). The rats in T1 group has lower serum kisspeptin and estradiol levels than those in T2 and C groups and smaller litter size of rats than those in the control group after mating (p < 0.05). No significant difference was observed between T2 and C groups. The levels of KISS1 and GPR54 mRNA in the hypothalamus and ovaries of the T1 group were significantly lower than that in control group. However, the levels of KISS1 mRNA in the T2 group were significantly lower than that in the control group only in ovaries (p < 0.05). CONCLUSION: The oral KISS1-54 DNA vaccine with fused tPA signal peptide was more effective than that KISS1-10 DNA vaccine in suppressing fertility of female rats.