Myostatin (MSTN), encoded by the MSTN gene, is a critical negative regulator of skeletal muscle mass. This study aims to identify and characterize the miRNAs involved in the development of the double-muscling phenotype in MSTN-deficient rabbits. We performed high-throughput sequencing to analyze the miRNA expression profiles in gluteus maximus tissue from wild type (MSTN+/+) and MSTN-KO (MSTN+/- and MSTN-/- inclusive) rabbits. Differentially expressed miRNAs (DEmiRNAs) were identified, and their potential target genes were predicted. Functional enrichment analysis of these target mRNAs was conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to elucidate the involved biological pathways and regulatory networks. A total of 25 DEmiRNAs (13 downregulated and 12 upregulated, |log2FC| ≥ 1.0, adjusted p < 0.05) and 1178 differentially expressed mRNAs (408 upregulated and 770 downregulated, |log2FC| ≥ 2.0, adjusted p < 0.05) were identified in MSTN-KO compared to MSTN+/+ rabbits. Bioinformatics analysis revealed that the target genes of these DEmiRNAs were significantly enriched in key pathways governing muscle growth and metabolism, including the PI3K-Akt signaling pathway, MAPK signaling pathway, and pathways related to ECM-receptor interaction and insulin signaling. Notably, many predicted target mRNAs are expressed by genes that encode key inhibitors of myogenesis (e.g., HDAC4) and major extracellular matrix components (e.g., COL4A3, POSTN). Our results demonstrate that MSTN deficiency induces a distinct and widespread change in the miRNA expression landscape of skeletal muscle.
A previous genome-wide association study identified regions on canine chromosome (cfa) 13 and 14 associated with early onset myxomatous mitral valve disease (MMVD) in Cavalier King Charles Spaniels (CKCS). In the present study, whole genome sequencing (WGS) of 9 CKCS cases (mitral regurgitation (MR) before 4.5 years or congestive heart failure (CHF) at any age due to MMVD) and 10 CKCS controls (no or mild MR after 8 years of age) identified > 2000 genetic variants in the MMVD associated cfa13 and cfa14 regions. Ensembl Variant Effect Predictor (VEP) identified a possible functional impact of 18 variants. These were genotyped in 250 CKCS; 117 cases and 133 controls. The most significantly associated variants were a splice-site variant in a long noncoding RNA (lncRNA) on cfa13, a nonsynonymous variant in HYAL4, a 39 base-pair insertion in LMOD2 and a synonymous variant in ENSCAFG00000024436 (p-values from 2.03E-08 to 4.20E-06). Concomitant homozygosity for risk alleles in LMOD2 and the lncRNA gave an odds-ratio for MMVD of 52.5 compared to homozygosity for the nonrisk alleles (p = 0.00034, 95% CI: 8.8-1023.8). Upon validation of our results in an independent cohort, this gene variant combination in CKCS is expected to enable targeted breeding programs to reduce MMVD prevalence in CKCS.
Wattles are finger-like appendages on the ventral neck of goats, serving as a distinctive morphological marker for breed identification that serves potential implications for production performance. However, their genetic basis remains incompletely characterized. Here, we integrated a genome-wide association study (GWAS) and selection signature analysis to identify candidate genes and genomic regions associated with the wattle trait in goats. Using a linear mixed model, GWAS on 463 goats (23 wattled and 440 non-wattled) identified 385 quantitative trait loci (QTLs) at a 5% false discovery rate, yielding 346 candidate genes. The most significant association signal was observed on chromosome 10 (72.61-73.48 Mb), where the lead SNP (rs636481767) is located within a region containing GJD2, GREM1, and FMN1, showing strong linkage disequilibrium (r2 > 0.6) with surrounding loci. Subsequent selection signature analysis (23 wattled and 23 non-wattled) identified 83 genomic regions harboring 119 candidate genes. The strongest signals were detected at MFSD14B on chromosome 8 (FST = 0.154, log2π-ratio = 2.611) and PDLIM7 on chromosome 7 (FST = 0.144, log2π-ratio = 0.806). KEGG pathway enrichment analysis revealed that GWAS-associated genes were involved in glycosylation and immune responses, whereas selection-signature genes were enriched in DNA repair and the Hippo, Notch, and Wnt pathways. Furthermore, cross-species PheWAS revealed that human FMN1 is associated with dermatological, skeletal, and metabolic phenotypes, while porcine FMN1 is associated with backfat thickness and loin muscle depth. Overall, this study provides molecular markers of potential value for goat breeding and pinpoints key candidate genes for future functional validation of wattle development.
Nutrigenomics investigates how nutrients modulate gene expression. Among them, fatty acids (FA) play important roles in regulating gene transcription, while long non-coding RNAs (lncRNAs) may be associated with gene regulation and metabolic diseases. This study aimed to analyze the hepatic transcriptome of pigs, a species frequently used as a model for nutrigenomic studies, to identify novel lncRNAs and their potential target genes in response to diets containing different sources of FA. Seventy-two pigs were fed four diets supplemented with 1.5% soybean oil (control), 3% canola oil, 3% fish oil, and 3% soybean oil. RNA sequencing of liver samples was performed to identify novel lncRNAs. Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify modules associated with phenotypic traits related to lipid metabolism and inflammation. Functional enrichment analyses were then conducted to annotate genes within these modules using Gene Ontology (GO) terms and to assess overlap with Quantitative Trait Loci (QTL). The results revealed 106 novel lncRNAs potentially regulating genes associated with lipid metabolism and immune responses in pigs fed diets with different FA sources. These findings enhance understanding of the regulatory role of lncRNAs in pigs and reinforce their relevance as models for human metabolic diseases.
The Min pig, a representative northern Chinese indigenous breed, carries a unique ancestral background shaped by the historical phylogeography of Northeast Asia. This study aimed to dissect the population structure, temporal genetic divergence, and ancestral composition of Min pigs, trace their evolutionary origin, and identify trait-linked functional genes, providing information regarding their evolutionary history and conservation. We analyzed 61 Min pigs sampled across nearly 20 years and 701 reference pigs comprising other Chinese indigenous breeds, Western commercial lines, and Chinese wild boars, using PCA, NJ phylogenetic analysis, Admixture, TreeMix, D-statistic, f4-ratio, and combined selection signature scans (sliding-window FST, XP-EHH, and π-ratio). Clear genetic stratification was observed among Min pig subpopulations, reflecting long-term divergence under natural and artificial selection. PCA and Admixture (K = 2-4) separated East Asian indigenous and Western ancestral components, verifying an admixed Northeast Asian origin with a dominant ancient East Asian component and a Western component. Compared with early-2000s Min pigs, contemporary individuals are genetically closer to Western breeds and exhibit a more scattered structure due to shifted ancestral component proportions, further confirmed by D-statistic and f4-ratio values. We identified 321 differentiated SNP loci based on the Animal QTL database, corresponding to core candidate genes (AKT3, ACACA, MAP3K5, FGFR4, C3, and SERPINC1) enriched for meat quality, growth, reproduction, immunity, energy metabolism, and MAPK/PI3K-Akt/AMPK pathways. This study reveals Min pigs' admixed origin and temporal divergence, clarifying their Northeast Asian evolution and providing molecular markers for genetic monitoring and conservation.
The Animal Variant Classification Guidelines (AVCG) were developed to standardize and objectify the classification of putative disease-causing variants. These guidelines are sufficiently reproducible and are used to classify previously published and new disease-causing variants across species. Here, the guidelines are updated (AVCG.v2), based on a three-phase decision process. Overall, four new criteria and seven clarifying comments were added. The number of criteria has increased from 23 to 27, with three new criteria supporting pathogenicity and one new criterion supporting benign classification. Pharmacogenomic variants were determined to fall within the scope of the guidelines. These updated guidelines are being used by the Variant Pathogenicity Working Group (VPWG), part of the Animal Genetic Testing Standardization standing committee, which is a committee of elected members of the International Society for Animal Genetics (ISAG). Under the auspices of ISAG, the VPWG retrospectively classifies published putative disease-causing variants. The pathogenicity label for a variant will be presented in the variant tables of Online Mendelian Inheritance in Animals (OMIA; https://omia.org/). The AVCGv.2 criteria and recommendations were developed by the expertise of the animal genetics community and the ISAG Executive Committee through the Animal Genetics Testing Standardization Committee endorses and strongly encourages their use to evaluate the evidence supporting pathogenicity of putative disease-causing variants.
This study presents a comparative analysis of the genetic structure and diversity of three red fox (Vulpes vulpes L.) populations representing different microevolutionary scenarios: panmixia (free-ranging Belarusian foxes), geographic isolation (free-ranging Scottish foxes), and anthropogenic selection (farm-bred foxes). Using a validated set of STR markers, multivariate statistical analysis was conducted to assess the genetic structure and the degree of genetic erosion across the studied groups. The wild red fox population in Belarus has been shown to maintain a state close to panmixia (PHWE = 0.090), characterized by a high effective population size (Ne = 694) and high allelic diversity. The island population from Scotland exhibits moderate gene pool depletion (Ne = 75.9) and a pronounced heterozygote deficiency (FIS = 0.18). Critical genetic erosion, which was characterized by a minimal effective population size (Ne = 60.2) and allelic fixation, was detected in the farm-bred group. The genetic distance between farm-bred and wild foxes (FST = 0.279; p = 0.001) reflects both the phylogeographic divergence between the Nearctic ancestors of farmed lineages and Palearctic wild populations, and the consequences of prolonged anthropogenic isolation, genetic drift, and selective breeding. These data indicate that artificial isolation and the impacts of genetic drift and targeted selection lead to a substantial depletion of the species' adaptive potential.
With the global population expected to reach 10 billion by 2050, sustainable livestock production is critical. Gene editing of the myostatin (MSTN) gene represents a promising strategy to enhance muscle growth in cattle. In this study, MSTN-mutated founder (F0) cows were used to generate F1 offspring via ovum pick-up, in vitro fertilization, and embryo transfer. Four F1 calves were born, all confirmed to be heterozygous for the MSTN mutation. Long-term monitoring showed normal growth and no visible health abnormalities. Whole-genome sequencing identified SNPs, INDELs, and structural variants, most with minimal predicted functional effects. Proteomic profiling of Longissimus dorsi muscle quantified 2947 proteins, revealing only subtle expression differences between MSTN-mutated and wild-type cattle. These results demonstrate stable inheritance and confirm that MSTN editing does not disrupt genome integrity or protein expression. Overall, our findings support the safety and utility of MSTN gene editing to improve livestock productivity for future food security.
Rex rabbit fur quality determines economic value, yet the age-dependent dynamics of hair follicle development and their molecular drivers are largely unknown. Here, we performed a time-series integrative analysis combining phenotypic measurements, quantitative histomorphology, and transcriptome sequencing in Yongqing Rex rabbits across 1 to 6 months of age (10 rabbits per month). Fur thickness exhibited a pattern of increase, stabilization, and subsequent increase, with no significant differences between 2M and 4M (p > 0.05). Coat density peaked at 6M, with an average pelt area of 1358.20 ± 52.43 cm2 at 5M and 6M. Fiber length increased and then plateaued, whereas hair diameter showed an increase-decrease-stabilization pattern. Histological analysis revealed a significant age-related decrease in the number of follicle clusters, particularly from 1M to 3M (p < 0.05). Primary hair follicle density was the highest at 1M (p < 0.01) and fluctuated subsequently, whereas secondary hair follicle density increased continuously (p < 0.01). Melanin granules increased significantly from 1M to 4M and peaked at the 4M (p < 0.05). Further, transcriptomic identified 1168, 4204, and 3431 differentially expressed genes (DEGs) for 3M versus 1M, 5M versus 1M, and 5M versus 3M, respectively, with 187 common DEGs including FOXN1, FGF10, and DCN. GO and KEGG analysis indicated significant enrichment in processes such as keratinization, keratinocyte differentiation, ECM-receptor interaction, and the PI3K-Akt signaling pathway. Collectively, our findings reveal the dynamic patterns of hair follicle development in Rex rabbits across phenotypic, histological, and molecular levels, providing a theoretical basis for the genetic regulation of fur quality.
In this study, we analysed genetic diversity, population structure and inbreeding in the Lipica Stud Farm population by integrating pedigree records, microsatellite (STR) and genome-wide SNP data generated using the GGP Equine 70K BeadChip. The dataset comprised 233 horses from the Lipica Stud Farm, which served as the reference population for comparisons of diversity estimates based on pedigree-, STR- and SNP data. The STR data were originally generated for routine parentage verification and were included here to compare inbreeding estimates based on pedigree, STR and genome-wide SNP data. Pedigrees of the analysed horses were traced back to the 18th century, with the longest ancestral path spanning 35 generations. The mean pedigree inbreeding coefficient in the reference population was 11.55%, while ancestral inbreeding coefficients ranged from 5.93% to 15.42%. Microsatellite analyses revealed an average observed heterozygosity (Ho) of 0.68 and a mean of 5.9 alleles per locus, with STR-based inbreeding (FSTR) ranging from 0.10 to 0.15. However, genomic inbreeding estimated from runs of homozygosity (FROH) varied between 0.09 and 0.28, with an average of 0.16. Selection signature analysis identified several putative candidate regions, whereas integrated haplotype score (iHS) analysis highlighted three candidate protein-coding genes (ZNF114, DENND5A, and TENM4). The ROH islands contained 24 genes previously associated with pigmentation, reproduction, muscle function, and other biologically relevant traits in horses. These results provide a comprehensive genomic characterisation of the Lipica Stud Farm population and offer important insights into genetic diversity and inbreeding patterns in the Lipizzan breed.
Wildlife forensic analysis frequently deals with highly degraded DNA samples, including those from tanned hides, processed products of traditional Chinese medicine, and specimens stored in preservation fluids. These samples present significant challenges for traditional DNA barcoding due to the difficulty of amplifying sufficiently long DNA fragments. To overcome this limitation, we designed five primer pairs to amplify short mitochondrial DNA (mtDNA) fragments (~100 base pairs). When aligned collectively, these fragments span approximately 500 bp of the cytochrome C oxidase subunit I (COI) gene, which is one of the most common species-barcoding targets. We subjected the primers to a comprehensive validation process, including in silico analysis and experimental verification using various non-degraded and degraded samples of animal tissues. Here, we demonstrate the efficacy and reliability of our DNA minibarcoding method. By addressing the limitations of traditional DNA barcoding, this method improves the accuracy and success of species identification, thereby supporting wildlife conservation, management, and forensic investigations.
We investigated a random-bred cat with sebaceous gland dysplasia. The cat presented with hypotrichosis and dark crusts on the skin. Histopathologically, sebaceous glands were enlarged and had an abnormal morphology with an increased number of undifferentiated reserve cells. Whole genome sequence analysis of the affected cat and comparison to 106 genomes of genetically diverse cats revealed a private homozygous nonsense variant in the functional candidate gene SOAT1 encoding sterol O-acyltransferase 1, XM_011291017.4:c.1221G>A or XP_011289319.1:p.(Trp407*). Based on the known role of SOAT1 in sebaceous gland function, the identified variant most likely represents the cause for the phenotype. To the best of our knowledge, we report the second pathogenic SOAT1 variant in cats. These results enable diagnostic genetic testing should any new cases come under veterinary care, detection of unaffected carrier animals, and strengthen the gene-phenotype relationship between SOAT1 and sebaceous gland dysplasias.
Eggshell color is an important economic trait in poultry. In green-eggshell chickens, eggshell color intensity is primarily determined by the amount of biliverdin deposited in the shell, but its genetic basis remains unclear. In this study, 230 Dongxiang green-eggshell chickens at 29 weeks of age were genotyped for the green-shell locus. Homozygous green-shell individuals were identified and subjected to a genome-wide association study (GWAS) using eggshell biliverdin concentration as the phenotype. The results showed that all 230 individuals were homozygous for the green-shell genotype, and eggshell biliverdin concentration was approximately normally distributed in the population. GWAS identified seven SNPs that exceeded the genome-wide significance threshold within a 110-kb region (3035804-3143838 bp) on chromosome 12. Functional annotation of the significant loci identified ALAS1 as a candidate gene. ALAS1 encodes the rate-limiting enzyme in heme biosynthesis and is therefore biologically linked to biliverdin production. Haplotype analysis further revealed a strong linkage block encompassing the ALAS1 region, supporting its potential role in regulating green eggshell color intensity. These findings provide candidate genetic markers for improving green eggshell color intensity in breeding programs.
Hematological adaptation is critical for oxygen transport at high altitudes. While hemoglobin concentration is well-studied, yak (Bos grunniens) hematology versus lowland cattle remains under-explored. We analyzed blood phenotypes from 1244 plateau yaks and 154 lowland cattle. Yaks exhibited elevated hemoglobin, hematocrit, and lymphocyte counts, but reduced platelet counts and monocyte/neutrophil percentages among other parameters, indicating distinct hypoxia adaptation. Using genome-wide association studies (GWAS) and composite of multiple signals (CMS), we identified 6485 selection-associated single-nucleotide variants (SNVs) linked to 43 hematological traits (average 6.85% variance explained). These included 172 yak-selected genes (YSGs), with SLIT3, FSTL5, and PCDH15 showing top pleiotropic effects (SLIT3 regulates immune function and oxygen transport). Thirty-eight YSGs influenced multiple traits, and six (SLIT3, FSTL5, PCDH15, PRIM2, CDH13, HLA-DQB1) overlap with genes under selection in Tibetan highlanders. Crucially, unlike hemoglobin attenuation in humans and other species, yaks show elevated hemoglobin, suggesting an alternative adaptive strategy. Cross-species analysis revealed 76 YSG orthologues affect 13 human blood traits, indicating highland species are likely to have selected the same set of genes for hypoxia adaptation. Our findings demonstrate polygenic, pleiotropic mechanisms underpinning yak adaptation, providing a genomic framework for high-altitude biology and conservation.
Primary ciliary dyskinesia (PCD) is a clinical syndrome that in dogs primarily manifests as chronic respiratory disease associated with cilial malfunction. The current study employs whole-genome sequencing and a candidate gene approach to uncover the genetic basis of PCD in three Cocker Spaniel siblings following diagnosis of their respiratory cilia by scanning electron microscopy and high-speed video microscopy. Absence of the disorder in the parents suggested autosomal recessive inheritance. A 29 bp frameshift insertion in the eleventh exon of the candidate gene sperm-associated antigen 1 (SPAG1) [NC_049234.1:g.2213788_2213789insGGCGGCGGCAAGCGGCCGGAGAGGGGCGC] was identified as likely causative for PCD in this family. The 29 bp frameshift variant was unobserved in a public variant call file including 1987 dogs from the Dog10K resource however an in-frame insertion was sometimes observed at the same locus. A Cocker Spaniel with similar symptoms from a different family tested negative for the identified variant suggesting that there are multiple causes for the condition in Cocker Spaniels.
Cold shock proteins (CSPs) play important roles in cellular adaptation to environmental stress. However, their characteristics and potential roles in yak (Bos grunniens), a species adapted to high-altitude environments, remain poorly understood. In this study, a genome-wide identification and comparative analysis of the CSP gene family was performed primarily using bioinformatics approaches based on publicly available genomic and transcriptomic datasets, along with a preliminary validation of their differential expression under cold and hypoxic stress. A total of 14 CSP genes were identified in the yak genome. Phylogenetic analysis across eight bovine species classified these genes into nine distinct clusters, revealing evolutionary conservation within the CSP family. Structural analyses showed variation in exon-intron organization and conserved motifs associated with stress responses. Protein-protein interaction (PPI) network analysis further suggested potential functional interactions between CSPs and key regulatory proteins. Tissue transcriptomic data indicated distinct expression patterns of CSP genes across multiple tissues. To provide experimental support for these findings, Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis was conducted in yak preadipocytes exposed to cold and hypoxic stress, revealing significant upregulation of several CSP genes. Together, these results provide a comprehensive overview of the CSP gene family in yak and offer insights into their potential roles in high-altitude adaptation in bovine species.
Rabbit meat is widely recognized for its favorable nutritional properties, while the molecular mechanisms underlying differences in meat quality between New Zealand White rabbits and Rex rabbits are essential for rabbit production. In this study, a total of 120 healthy 13-week-old rabbits (60 per group) were used to evaluate meat quality traits. The longissimus dorsi muscle (LDM) was then collected for transcriptomic and metabolomic analyses. The results showed that New Zealand White rabbits exhibited significantly higher live weight before slaughter, eviscerated weight, semi-eviscerated weight, and pH24 (p < 0.01), whereas Rex rabbits displayed higher cooking loss (p < 0.01) and intramuscular fat content (p < 0.05). Metabolomic profiling identified 218 differential metabolites (DMs), which were mainly enriched in amino acid biosynthesis and the pentose phosphate pathway, including key metabolites such as DL-arginine, gallic acid, and lipid-related compounds. Transcriptomic analysis identified 227 differentially expressed genes (DEGs) enriched in pathways associated with muscle development and meat quality, including oxidative phosphorylation, FoxO, and MAPK signaling pathways. Key DEGs, such as MYH13, HOXA13, and PDK4, were associated with muscle fiber formation and fat deposition. Integrated analysis revealed that 34 DEGs and 24 DMs were co-enriched in 26 pathways, with strong correlations observed between oxidative phosphorylation-related genes and energy metabolites, as well as between collagen-associated genes and amino acids. These findings establish a gene-metabolite regulatory network underlying breed-specific differences in meat quality and identify potential molecular markers to improve rabbit meat quality and to better understand muscle metabolism across different rabbit breeds.
The commercial pork production sector prioritizes genetic improvements in lean meat percentage to enhance profitability and meet consumer preferences. The Pietrain pig, a premier terminal sire breed renowned for its exceptional muscularity and leanness, serves as an ideal model to decipher the genetic underpinnings of these traits. This study investigated two key measures of leanness, backfat thickness and loin muscle depth, in two distinct Pietrain populations to elucidate the genetic architecture underlying these traits. We estimated genetic parameters and performed a meta-analysis of genome-wide association studies, identifying ABCD4, LTBP2, NUMB, and SLC30A9 as candidate genes. To further investigate these associations, we integrated information on molecular quantitative trait loci from the PigGTEx project and single-cell transcriptomic resources. This integrative approach prioritized ABCD4 as a key candidate gene regulating backfat thickness. Functional validation in 3T3-L1 preadipocytes revealed a novel dual regulatory role for ABCD4: its knockdown suppressed cell proliferation while simultaneously stimulating adipogenic differentiation, as demonstrated by the upregulation of key markers. Our findings positioned ABCD4 as a critical modulator of fat deposition, likely through its influence on the core adipogenic transcriptional network. By establishing an analytical framework that integrates large-scale sequencing data from Pietrain pigs with functional validation, our study addresses a key gap in understanding the genetic basis of leanness and provides novel insights for precision breeding.
Canine mammary tumours (CMTs) are one of the most frequently observed malignancies in female dogs. Many studies have investigated epigenetic changes in CMT at the nuclear level, yet the role of mtDNA methylation, particularly in the D-loop region, remains largely unexplored. Given the significant importance of the D-loop region in mtDNA replication and transcription, and the previous identification of CpG enrichment there, this preliminary study aimed to assess apparent methylation signals in the mitochondrial D-loop region. We examined CpG, CHG and CHH methylation contexts in matched tumour and normal tissues from six German Shepherd dogs with histologically classified mammary carcinomas using enzymatic methyl-sequencing and high-throughput amplicon sequencing. Overall, CpG and CHH contexts exhibited greater variability between tumour and normal tissues than CHG, which remained relatively stable. Correlation analyses revealed a putative positive relationship between differences in CpG and CHH methylation (Spearman's r = 0.829, p = 0.042). This might suggest a coordinated epigenetic modulation of these two methylation patterns in the carcinogenesis process, yet due to the limited study group (n = 6) the hypothesis should be verified on a larger cohort. These findings demonstrate a plausible existence of intra-individual epigenetic divergence at the mitochondrial level. To our knowledge, this is the first study to assess the influence of mtDNA methylation on CMT, and it supports further investigation of mitochondrial epigenetics in canine oncology.
