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. 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. 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). 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.

Background: There are abundant sheep breed resources in the Xinjiang region of China attributing to its diverse ecological system, which include several high-litter size sheep populations. Previous studies have confirmed that the major high prolificacy gene cannot be used to detect high litter size. Our research team found a resource group in Pishan County, southern Xinjiang. It showed high fertility with an average litter size of two to four in one birth, excellent breast development, and a high survival rate of lambs. In the present study, we used this resource as an ideal sample for studying the genetic mechanisms of high prolificacy in sheep. Methods: Indigenous sheep populations from Xinjiang, with different litter sizes, were selected for the research, and specific-locus amplified fragment sequencing (SLAF-seq) technology was used to comprehensively screen single nucleotide polymorphisms (SNPs) from the whole genome that may cause differences in litter size. Novel genes associated with litter size of sheep were detected using genome-wide association studies (GWAS), providing new clues revealing the regulation mechanism of sheep fecundity. Candidate genes related to ovulation and litter size were selected for verification using Kompetitive Allele Specific polymerase chain reaction (KASP) cluster analysis. Results: We identified 685,300 SNPs using the SLAF-seq technique for subsequent genome-wide analysis. Subsequently, 155 SNPs were detected at the genome-wide level. Fourteen genes related to sheep reproduction were notated: COIL, SLK, FSHR, Plxna3, Ddx24, CXCL12, Pla2g7, ATP5F1A, KERA, GUCY1A1, LOC101107541, LOC101107119, LOC101107809, and BRAF. Based on literature reports, 30 loci of seven genes and candidate genes (CXCL12, FSHR, SLK, GUCY1A1, COIL, LOC101107541, and LOC101107119) related to ovulation and litter size were selected for verification using KASP cluster analysis. Among them, nine loci of three genes were successfully genotyped. Three loci of FSHR (GenBank ID: 443299, g. 75320741G>A site), GUCY1A1 (GenBank ID: 101110000, g. 43266624C>T site), and COIL (GenBank ID: 101123134, g. 7321466C>G site) were found to be significantly or extremely significantly associated with litter size. These three loci are expected to be used as molecular markers to determine differences in litter size in sheep.

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5′ RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region −405 to −200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5’ regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

Residual feed intake (RFI) is a key indicator of feed efficiency in ruminants. To elucidate the potential regulatory roles of microorganisms and metabolites under different RFI levels, we investigated 24 polytocous fine-wool sheep (12 high-RFI and 12 low-RFI) using metagenomic sequencing and non-targeted metabolomics of rumen and rectal contents. Significant differences in average daily feed intake, residual feed intake, and feed conversion ratio were observed between groups ( p < 0.001). LEfSe analysis identified four and seventeen RFI-associated microbial biomarkers in the rumen and rectum, respectively, with s_Ruminococcus_albus and s_Ruminococcus_bicirculans as common core taxa. Functional annotation revealed that high-RFI sheep were enriched in amino acid metabolism and xenobiotic degradation pathways in the rumen, whereas low-RFI sheep were enriched in pathways related to development and regeneration. In the rectum, high-RFI sheep showed enrichment in protein folding and degradation, carbohydrate metabolism, and energy metabolism, while low-RFI sheep were enriched in transcriptional regulation and signal transduction pathways. Metabolomic analysis detected 297 and 1,130 differential metabolites in the rumen and rectum, respectively, mainly lipids, organic acids, and derivatives. KEGG enrichment indicated that rumen metabolites were primarily involved in bile acid biosynthesis and riboflavin metabolism, while rectal metabolites were enriched in energy metabolism and multiple amino acid pathways, including arachidonic acid, tryptophan, tyrosine, lysine, and methionine metabolism. Integrated analysis revealed significant associations between key bacterial taxa and metabolites, and network construction identified core nodes potentially engaged in synergistic regulation, providing insights into their roles in RFI phenotype formation. Collectively, these findings highlight the distinct contributions of the rumen and rectum to feed efficiency in sheep and offer theoretical support for nutritional regulation strategies to improve ruminant production performance.

Introduction: Mutton has the advantages of delicious taste, high nutrition, and easy digestion. It is important to improve the production and quality of mutton in mutton sheep breeding. Microsatellite locus marker-assisted breeding is widely used to breed excellent traits of various species. It is important to search for microsatellite markers related to the economic traits (mutton production and fat content) of mutton sheep.Methods: This study aimed to explore the relationship between 11 microsatellite loci of Texel × Kazakh sheep and 12 economic traits and to seek potential loci related to the mutton production (PW: Pre-slaughter weight, CW: Carcass weight, TAW: Total breast and abdomen weight, TLT: Total weight of left anterior tendon, TLL: Total weight of left hip and leg, LD: Longissimus dorsi, OMA: Ocular muscle area) and fat deposition levels (TFW: Tail fat weight, MFW: Mesenteric fat weight, KFW: Kidney fat weight, BFT: Back fat thickness and GR: GR value) of mutton sheep.Results: Genetic analysis of the 108 Texel × Kazakh sheep hybrid population revealed 81 alleles across all loci, with a mean number of alleles (MNA) of 7.364. The population exhibited moderate observed heterozygosity (Ho = 0.610), high expected heterozygosity (He = 0.785), and substantial polymorphism (polymorphism information content, PIC = 0.759), indicating robust genetic diversity. Notably, the AMEL locus demonstrated significant associations with MFW (η² = 0.319) and KFW (η² = 0.347), while the INRA023 locus influenced CW (η² = 0.260) (adjusted p &lt; 0.05). No other loci showed statistically significant trait correlations after multiple-testing correction. The HH genotype at AMEL and AD genotype at INRA023 emerged as pivotal molecular markers, collectively explaining 26.0–34.7% of phenotypic variance in meat yield traits.Discussion: These findings establish a theoretical framework for precision breeding strategies, offering actionable solutions to enhance meat productivity in ovine populations through marker-assisted selection (MAS).