Interspecific hybridization often shows negative effects on hybrids. However, only a few multicellular species, limited to a handful of plants and animals, have shown partial genetic mechanisms by which hybridization leads to low fitness in hybrids. Here, to explore the outcome of combining the two genomes of a horse and donkey, we analyzed the whole-genome sequences from an Equus parent-offspring trio using Illumina platforms. We generated 41.39× and 46.21× coverage sequences for the horse and mule, respectively. For the donkey, a 40.38× coverage sequence was generated and stored in our laboratory. Approximately 24.86 million alleles were discovered that varied from the reference genome. Single nucleotide polymorphisms were used as polymorphic markers for assigning alleles to their parental genomic inheritance. We identified 25,703 Mendelian inheritance error single nucleotide polymorphisms in the mule genome that were not inherited from the parents through Mendelian inheritance. A total of 555 de novo single nucleotide polymorphisms were also identified. The rate of de novo single nucleotide polymorphisms was 2.21 × 10⁻⁷ in the mule from the Equus parent-offspring trio. This rate is obviously higher than the natural mutation rate for Equus, which is also consistent with the previous hypothesis that interracial crosses may have a high mutation rate. The genes associated with these single nucleotide polymorphisms are mainly involved in immune processes, DNA repair, and cancer processes. The results of the analysis of three genomes from an Equus parent-offspring trio improved our knowledge of the consequences of the integration of parental genomes in mules.

Muscle fibers, as the fundamental units of muscle tissue, play a crucial role in determining skeletal muscle function through their growth, development, and composition. To investigate changes in muscle fiber types and their regulatory mechanisms in Mongolian horses (MG), Xilingol horses (XL), and Grassland-Thoroughbreds (CY), we conducted histological and bioinformatic analyses on the gluteus medius muscle of these three horse breeds. Immunofluorescence analysis revealed that Grassland-Thoroughbreds had the highest proportion of fast-twitch muscle fibers at 78.63%, while Mongolian horses had the lowest proportion at 57.54%. Whole-transcriptome analysis identified 105 differentially expressed genes (DEGs) in the CY vs. MG comparison and 104 DEGs in the CY vs. XL comparison. Time-series expression profiling grouped the DEGs into eight gene sets, with three sets showing significantly up-regulated or down-regulated expression patterns (p < 0.05). Additionally, 280 differentially expressed long non-coding RNAs (DELs) were identified in CY vs. MG, and 213 DELs were identified in CY vs. XL. A total of 32 differentially expressed microRNAs (DEMIRs) were identified in CY vs. MG, while 44 DEMIRs were found in CY vs. XL. Functional enrichment analysis indicated that the DEGs were significantly enriched in essential biological processes, such as actin filament organization, muscle contraction, and protein phosphorylation. KEGG pathway analysis showed their involvement in key signaling pathways, including the mTOR signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. Furthermore, functional variation-based analyses revealed associations between non-coding RNAs and mRNAs, with some non-coding RNAs targeting genes potentially related to muscle function regulation. These findings provide valuable insights into the molecular basis for the environmental adaptability, athletic performance, and muscle characteristics in horses, offering new perspectives for the breeding of Grassland-Thoroughbreds.

Colitis in equines has high morbidity and mortality rates, which severely affects the development of the equine-breeding industry. With the issuance of antibiotic bans, there is an urgent need for healthier and more effective alternatives. In recent years, probiotics have been widely used as microbial feed additives in animal husbandry, playing a crucial role in preventing and treating diarrhea and regulating host immune function. In this study, we isolated and screened a strain with rapid and stable acid production using bromocresol purple, litmus milk coloration tests, and acid production performance assessments. Based on morphological characteristics, physiological and biochemical properties, and 16S rDNA identification, the strain was identified as Pediococcus pentosaceus and named M6. The Pediococcus pentosaceus M6 exhibited stable growth and tolerance to high temperatures, acid and bile salt concentrations, and simulated gastrointestinal fluid environments. The M6 strain demonstrated good antibacterial effects against Escherichia coli, Staphylococcus aureus, and Salmonella. The M6 strain did not produce hemolysis zones on Columbia blood agar plates, indicating its high safety, and was found to be insensitive to 12 antibiotics, including cephalexin and neomycin. Additionally, intervention in mice with dextran sulfate sodium (DSS)-induced colitis alleviated weight loss and shortened colon length. To a certain extent, it regulated the expression of inflammatory cytokines and the gut microbiota within the body and reduced inflammatory cell infiltration and intestinal barrier damage. In summary, the isolated Pediococcus pentosaceus M6 strain exhibited excellent probiotic properties and could alleviate DSS-induced colitis in mice, suggesting its potential application value as a probiotic in animal husbandry.

Speed and endurance are the primary goals in racehorse breeding. The Grassland-Thoroughbred is a newly developed breed in northern China that combines speed, endurance, and environmental adaptability. However, current research on the genetic background of this breed and the genes associated with athletic performance remains limited. We conducted whole-genome resequencing on Mongolian (MG), Thoroughbred (TB), Xilingol (XL), and Grassland-Thoroughbred (CY) horses, generating 3813.74 Gb of clean data after quality control. The number of transitions was significantly higher than that of transversions. The SNPs were mainly located in intergenic regions, followed by intronic regions. Principal component analysis, population structure analysis, and phylogenetic tree results indicated that the CYs had a distinct genetic background from MGs, TBs, and XLs, but based on PCA and phylogenetic clustering, they showed greater genetic similarity to Thoroughbreds. Using fixation index (Fst) and nucleotide diversity ratio (π ratio) analyses between CYs and the other three horse populations, 70, 76, and 80 candidate genes were identified from the intersection of the two methods, respectively. A total of 179 candidate genes were obtained from the union of the three groups. Candidate genes associated with athletic performance (ATF2, NDUFS7, PRKG1, IGFN1, MTOR, TTN) and growth and development (MTOR, IGFN1, COL21A1, NEDD4, PIEZO1) were screened. These genes are related to athletic ability and developmental processes in the CY population. Our study reveals genomic information associated with important traits in Grassland-Thoroughbreds and identifies valuable candidate genes, laying a foundation for future breeding and trait association studies.

The core objective of racehorse breeding is to enhance the speed and endurance of the horses. The Grassland-Thoroughbred is an emerging horse breed developed in northern China in recent years, characterized by excellent speed performance, enduring stamina, and strong environmental adaptability. However, research on the genetic characteristics within this breed and the genes associated with athletic performance remains relatively limited. We conducted whole-genome resequencing of Grassland-Thoroughbred F1, F2, F3, and the crossbred population (CY) and obtained a total of 4056.23 Gb of high-quality data after quality control. The single nucleotide polymorphisms (SNPs) were primarily distributed in intergenic regions, followed by intronic regions. Principal component analysis (PCA) and STRUCTURE revealed clear distinctions among the generations, with a notable overlap between CY and F3. Using the SNP dataset, we analyzed the number and length distribution patterns of runs of homozygosity (ROHs) in the genomes of different generational groups of Grassland-Thoroughbreds. Short ROHs ranging from 0.5 to 2 Mb were the most abundant, with the following distribution: F1 (85.15%) > F2 (82.92%) > CY (78.75%) > F3 (77.51%). Medium-length ROHs (2–8 Mb) and long ROHs (>8 Mb) together exhibited a similar but opposite trend. The average length of ROHs was 1.57 Mb. The inbreeding coefficients (F_ROH) among different generational groups of Grassland-Thoroughbreds were as follows: F1 (0.0942) < F2 (0.1197) < CY (0.1435) < F3 (0.1497). Through ROH island analysis, 10 high-frequency ROH regions were identified and annotated with 120 genes. Genomic regions and candidate genes associated with athletic traits—ACAD8, OPCML, PRDX2, NTM, NDUFB7, SCL25A15, FOXO1, and SLC4A10—were identified. These genes may play important roles in regulating muscle performance, mitochondrial energy supply, and learning and memory processes in horses and are closely associated with the athletic ability of the Grassland-Thoroughbred population. This study is the first to systematically characterize the genomic diversity and inbreeding dynamics of the Grassland-Thoroughbred during the breeding process. It identifies candidate genes that may influence athletic performance, thereby providing an important molecular foundation and theoretical basis for the genetic improvement and performance-based selection of this emerging breed.

Speed is not only the primary objective of racehorse breeding but also a crucial indicator for evaluating racehorse performance. This study investigates a newly developed racehorse breed in China. Through whole-genome resequencing, we selected 60 offspring obtained from the crossbreeding of Thoroughbred horses and Xilingol horses for this study. This breed is tentatively named “Grassland-Thoroughbred”, and the samples were divided into two groups based on racing ability: 30 racehorses and 30 non-racehorses. Based on whole-genome sequencing data, the study achieved an average sequencing depth of 25.63×. The analysis revealed strong selection pressure on chromosomes (Chr) 1 and 3. Selection signals were detected using methods such as the nucleotide diversity ratio (π ratio), integrated haplotype score (iHS), fixation index (Fst), and cross-population extended haplotype homozygosity (XP-EHH). Regions ranked in the top 5% by at least three methods were designated as candidate regions. This approach detected 215 candidate genes. Additionally, the Fst method was employed to detect Indels, and the top 1% regions detected were considered candidate regions, covering 661 candidate genes. Functional enrichment analysis of the candidate genes suggests that pathways related to immune regulation, neural signal transmission, muscle contraction, and energy metabolism may significantly influence differences in performance. Among these identified genes, PPARGC1A, FOXO1, SGCD, FOXP2, PRKG1, SLC25A15, CKMT2, and TRAP1 play crucial roles in muscle function, metabolism, sensory perception, and neurobiology, indicating their key significance in shaping racehorse phenotypes. This study not only enhances understanding of the molecular mechanisms underlying racehorse speed but also provides essential theoretical and practical references for the molecular breeding of Grassland-Thoroughbreds.