Genomic analyses of withers height and linear conformation traits in German Warmblood horses using imputed sequence-level genotypes.

BACKGROUND: Body conformation, including withers height, is a major selection criterion in horse breeding and is associated with other important traits, such as health and performance. However, little is known about the genomic background of equine conformation. Therefore, the aim of this study was to use imputed sequence-level genotypes from up to 4891 German Warmblood horses to identify genomic regions associated with withers height and linear conformation traits. Furthermore, the traits were genetically characterised and putative causal variants for withers height were detected. RESULTS: A genome-wide association study (GWAS) for withers height confirmed the presence of a previously known quantitative trait locus (QTL) on Equus caballus (ECA) chromosome 3 close to the LCORL/NCAPG locus, which explained 16% of the phenotypic variance for withers height. An additional significant association signal was detected on ECA1. Further investigations of the region on ECA3 identified a few promising candidate causal variants for withers height, including a nonsense mutation in the coding sequence of the LCORL gene. The estimated heritability for withers height was 0.53 and ranged from 0 to 0.34 for the conformation traits. GWAS identified significantly associated variants for more than half of the investigated conformation traits, among which 13 showed a peak on ECA3 in the same region as withers height. Genetic parameter estimation revealed high genetic correlations between these traits and withers height for the QTL on ECA3. CONCLUSIONS: The use of imputed sequence-level genotypes from a large study cohort led to the discovery of novel QTL associated with conformation traits in German Warmblood horses. The results indicate the high relevance of the QTL on ECA3 for various conformation traits, including withers height, and contribute to deciphering causal mutations for body size in horses.

Cows with diverging haplotypes show differences in differential milk cell count, milk parameters and vaginal temperature after S. aureus challenge but not after E. coli challenge.

BACKGROUND: In dairy cattle, mastitis causes high financial losses and impairs animal well-being. Genetic selection is used to breed cows with reduced mastitis susceptibility. Techniques such as milk cell flow cytometry may improve early mastitis diagnosis. In a highly standardized in vivo infection model, 36 half-sib cows were selected for divergent paternal Bos taurus chromosome 18 haplotypes (Q vs. q) and challenged with Escherichia coli for 24 h or Staphylococcus aureus for 96 h, after which the samples were analyzed at 12 h intervals. Vaginal temperature (VT) was recorded every three minutes. The objective of this study was to compare the differential milk cell count (DMCC), milk parameters (fat %, protein %, lactose %, pH) and VT between favorable (Q) and unfavorable (q) haplotype cows using Bayesian models to evaluate their potential as improved early indicators of differential susceptibility to mastitis. RESULTS: After S. aureus challenge, compared to the Q half-sibship cows, the milk of the q cows exhibited higher PMN levels according to the DMCC (24 h, p < 0.001), a higher SCC (24 h, p < 0.01 and 36 h, p < 0.05), large cells (24 h, p < 0.05) and more dead (36 h, p < 0.001) and live cells (24 h, p < 0.01). The protein % was greater in Q milk than in q milk at 0 h (p = 0.025). In the S. aureus group, Q cows had a greater protein % (60 h, p = 0.048) and fat % (84 h, p = 0.022) than q cows. Initially, the greater VT of S. aureus-challenged q cows (0 and 12-24 h, p < 0.05) reversed to a lower VT in q cows than in Q cows (48-60 h, p < 0.05). Additionally, the following findings emphasized the validity of the model: in the S. aureus group all DMCC subpopulations (24 h-96 h, p < 0.001) and in the E. coli group nearly all DMCC subpopulations (12 h-24 h, p < 0.001) were higher in challenged quarters than in unchallenged quarters. The lactose % was lower in the milk samples of E. coli-challenged quarters than in those of S. aureus-challenged quarters (24 h, p < 0.001). Between 12 and 18 h, the VT was greater in cows challenged with E. coli than in those challenged with S. aureus (3-h interval approach, p < 0.001). CONCLUSION: This in vivo infection model confirmed specific differences between Q and q cows with respect to the DMCC, milk component analysis results and VT results after S. aureus inoculation but not after E. coli challenge. However, compared with conventional milk cell analysis monitoring, e.g., the global SCC, the DMCC analysis did not provide refined phenotyping of the pathogen response.

Establishment of a cloning-free CRISPR/Cas9 protocol to generate large deletions in the bovine MDBK cell line.

The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.

Cryptosporidium parvum infection alters the intestinal mucosa transcriptome in neonatal calves: implications for immune function.

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies.

Sequence-based GWAS meta-analyses for beef production traits.

BACKGROUND: Combining the results of within-population genome-wide association studies (GWAS) based on whole-genome sequences into a single meta-analysis (MA) is an accurate and powerful method for identifying variants associated with complex traits. As part of the H2020 BovReg project, we performed sequence-level MA for beef production traits. Five partners from France, Switzerland, Germany, and Canada contributed summary statistics from sequence-based GWAS conducted with 54,782 animals from 15 purebred or crossbred populations. We combined the summary statistics for four growth, nine morphology, and 15 carcass traits into 16 MA, using both fixed effects and z-score methods. RESULTS: The fixed-effects method was generally more informative to provide indication on potentially causal variants, although we combined substantially different traits in each MA. In comparison with within-population GWAS, this approach highlighted (i) a larger number of quantitative trait loci (QTL), (ii) QTL more frequently located in genomic regions known for their effects on growth and meat/carcass traits, (iii) a smaller number of genomic variants within the QTL, and (iv) candidate variants that were more frequently located in genes. MA pinpointed variants in genes, including MSTN, LCORL, and PLAG1 that have been previously associated with morphology and carcass traits. We also identified dozens of other variants located in genes associated with growth and carcass traits, or with a function that may be related to meat production (e.g., HS6ST1, HERC2, WDR75, COL3A1, SLIT2, MED28, and ANKAR). Some of these variants overlapped with expression or splicing QTL reported in the cattle Genotype-Tissue Expression atlas (CattleGTEx) and could therefore regulate gene expression. CONCLUSIONS: By identifying candidate genes and potential causal variants associated with beef production traits in cattle, MA demonstrates great potential for investigating the biological mechanisms underlying these traits. As a complement to within-population GWAS, this approach can provide deeper insights into the genetic architecture of complex traits in beef cattle.

Improving the annotation of the cattle genome by annotating transcription start sites in a diverse set of tissues and populations using Cap Analysis Gene Expression sequencing.

Understanding the genomic control of tissue-specific gene expression and regulation can help to inform the application of genomic technologies in farm animal breeding programs. The fine mapping of promoters [transcription start sites (TSS)] and enhancers (divergent amplifying segments of the genome local to TSS) in different populations of cattle across a wide diversity of tissues provides information to locate and understand the genomic drivers of breed- and tissue-specific characteristics. To this aim, we used Cap Analysis Gene Expression (CAGE) sequencing, of 24 different tissues from 3 populations of cattle, to define TSS and their coexpressed short-range enhancers (<1 kb) in the ARS-UCD1.2_Btau5.0.1Y reference genome (1000bulls run9) and analyzed tissue and population specificity of expressed promoters. We identified 51,295 TSS and 2,328 TSS-Enhancer regions shared across the 3 populations (dairy, beef-dairy cross, and Canadian Kinsella composite cattle from 2 individuals, 1 of each sex, per population). Cross-species comparative analysis of CAGE data from 7 other species, including sheep, revealed a set of TSS and TSS-Enhancers that were specific to cattle. The CAGE data set will be combined with other transcriptomic information for the same tissues to create a new high-resolution map of transcript diversity across tissues and populations in cattle for the BovReg project. Here we provide the CAGE data set and annotation tracks for TSS and TSS-Enhancers in the cattle genome. This new annotation information will improve our understanding of the drivers of gene expression and regulation in cattle and help to inform the application of genomic technologies in breeding programs.

Replacement of microsatellite markers by imputed medium-density SNP arrays for parentage control in German warmblood horses.

In horses, parentage control is currently performed based on an internationally standardized panel of 17 microsatellite (MS) markers comprising 12 mandatory and five optional markers. Unlike MS, single nucleotide polymorphism (SNP) profiles support a wider portfolio of genomic applications, including parentage control. A transition to SNP-based parentage control is favorable, but requires additional efforts for ensuring generation-overlapping availability of marker genotypes of the same type. To avoid double genotyping of either parents or offspring for changing to SNP technology and enable efficient transition, we tested whether MS genotypes used for parentage control could be reliably imputed from a medium-density SNP panel in German warmblood horses. Imputation accuracy was tested in a tenfold cross-validation with two approaches: within breed (option A) and across breeds (option B). Average imputation accuracies of 97.98% (A) and 96.17% (B) were achieved, respectively. Due to interbreed differences in genotyping rates, five MS markers of low genotyping rate (GTR; < 90%) could be imputed with higher accuracy within breed (98.18%) than across breeds (90.73%). MS markers with high GTR performed homogeneously well in option B (98.44%) and showed slightly lower accuracy in option A (97.90%). Among these markers, AHT5 proved to be problematic for imputation regardless of the approach, revealing accuracies of 86.40% (A) and 88.70% (B). Better results for MS markers with high GTR and savings in computational processing justified the choice of option B for routine implementation. To date, more than 9500 horses have undergone the new parentage control based on imputed MS genotypes.

Allele-biased expression of the bovine APOB gene associated with the cholesterol deficiency defect suggests cis-regulatory enhancer effects of the LTR retrotransposon insertion.

The insertion of an endogenous retroviral long terminal repeat (LTR) sequence into the bovine apolipoprotein B (APOB) gene is causal to the inherited genetic defect cholesterol deficiency (CD) observed in neonatal and young calves. Affected calves suffer from developmental abnormalities, symptoms of incurable diarrhoea and often die within weeks to a few months after birth. Neither the detailed effects of the LTR insertion on APOB expression profile nor the specific mode of inheritance nor detailed phenotypic consequences of the mutation are undisputed. In our study, we analysed German Holstein dairy heifers at the peak of hepatic metabolic load and exposed to an additional pathogen challenge for clinical, metabolic and hepatic transcriptome differences between wild type (CDF) and heterozygote carriers of the mutation (CDC). Our data revealed that a divergent allele-biased expression pattern of the APOB gene in heterozygous CDC animals leads to a tenfold higher expression of exons upstream and a decreased expression of exons downstream of the LTR insertion compared to expression levels of CDF animals. This expression pattern could be a result of enhancer activity induced by the LTR insertion, in addition to a previously reported artificial polyadenylation signal. Thus, our data support a regulatory potential of mobile element insertions. With regard to the phenotype generated by the LTR insertion, heterozygote CDC carriers display significantly differential hepatic expression of genes involved in cholesterol biosynthesis and lipid metabolism. Phenotypically, CDC carriers show a significantly affected lipomobilization compared to wild type animals. These results reject a completely recessive mode of inheritance for the CD defect, which should be considered for selection decisions in the affected population. Exemplarily, our results illustrate the regulatory impact of mobile element insertions not only on specific host target gene expression but also on global transcriptome profiles with subsequent biological, functional and phenotypic consequences in a natural in-vivo model of a non-model mammalian organism.

From the comparative study of a circRNA originating from an mammalian ATXN2L intron to understanding the genesis of intron lariat-derived circRNAs.

Circular intronic RNAs (ciRNAs) are still unexplored regarding mechanisms for their emergence. We considered the ATXN2L intron lariat-derived circular RNA (ciRNA-ATXN2L) as an opportunity to conduct a cross-species examination of ciRNA genesis. To this end, we investigated 207 datasets from 4 tissues and from 13 mammalian species. While in eight species, ciRNA-ATXN2L was never detected, in pigs and rabbits, ciRNA-ATXN2L was expressed in all tissues and sometimes at very high levels. Bovine tissues were an intermediate case and in macaques and cats, only ciRNA-ATXN2L traces were detected. The pattern of ciRNA-ATXN2L restricted to only five species is not related to a particular evolution of intronic sequences. To empower our analysis, we considered 221 additional introns including 80 introns where a lariat-derived ciRNA was previously described. The primary driver of micro-ciRNA genesis (< 155 nt as ciRNA-ATXN2L) appears to be the absence of a canonical "A" (i.e. a "tnA" located in the usual branching region) to build the lariat around this adenosine. The balance between available "non canonical-A" (no ciRNA genesis) and "non-A" (ciRNA genesis) for use as a branch point to build the lariat could modify the expression level of ciRNA-ATXN2L. In addition, the rare localization of the 2'-5' bond in an open RNA secondary structure could also negatively affect the lifetime of ciRNAs (macaque ciRNA-ATXN2L). Our analyses suggest that ciRNA-ATXN2L is likely a functionless splice remnant. This study provides a better understanding of the ciRNAs origin, especially drivers for micro ciRNA genesis.

Genetic and genomic characterization followed by single-step genomic evaluation of withers height in German Warmblood horses.

Reliability of genomic predictions is influenced by the size and genetic composition of the reference population. For German Warmblood horses, compilation of a reference population has been enabled through the cooperation of five German breeding associations. In this study, preliminary data from this joint reference population were used to genetically and genomically characterize withers height and to apply single-step methodology for estimating genomic breeding values for withers height. Using data on 2113 mares and their genomic information considering about 62,000 single nucleotide polymorphisms (SNPs), analysis of the genomic relationship revealed substructures reflecting breed origin and different breeding goals of the contributing breeding associations. A genome-wide association study confirmed a known quantitative trait locus (QTL) for withers height on equine chromosome (ECA) 3 close to LCORL and identified a further significant peak on ECA 1. Using a single-step approach with a combined relationship matrix, the estimated heritability for withers height was 0.31 (SE = 0.08) and the corresponding genomic breeding values ranged from - 2.94 to 2.96 cm. A mean reliability of 0.38 was realized for these breeding values. The analyses of withers height showed that compiling a reference population across breeds is a suitable strategy for German Warmblood horses. The single-step method is an appealing approach for practical genomic prediction in horses, because not many genotypes are available yet and animals without genotypes can by this way directly contribute to the estimation system.

Metabogenomic analysis to functionally annotate the regulatory role of long non-coding RNAs in the liver of cows with different nutrient partitioning phenotype.

Long non-coding RNAs (lncRNAs) hold gene regulatory potential, but require substantial further functional annotation in livestock. Applying two metabogenomic approaches by combining transcriptomic and metabolomic analyses, we aimed to identify lncRNAs with potential regulatory function for divergent nutrient partitioning of lactating crossbred cows and to establish metabogenomic interaction networks comprising metabolites, genes and lncRNAs. Through correlation analysis of lncRNA expression with transcriptomic and metabolomic data, we unraveled lncRNAs that have a putative regulatory role in energy and lipid metabolism, the urea and tricarboxylic acid cycles, and gluconeogenesis. Especially FGF21, which correlated with a plentitude of differentially expressed genes, differentially abundant metabolites, as well as lncRNAs, suggested itself as a key metabolic regulator. Notably, lncRNAs in close physical proximity to coding-genes as well as lncRNAs with natural antisense transcripts appear to perform a fine-tuning function in gene expression involved in metabolic pathways associated with different nutrient partitioning phenotypes.

Single-cell RNA sequencing of freshly isolated bovine milk cells and cultured primary mammary epithelial cells.

Bovine mammary function at molecular level is often studied using mammary tissue or primary bovine mammary epithelial cells (pbMECs). However, bulk tissue and primary cells are heterogeneous with respect to cell populations, adding further transcriptional variation in addition to genetic background. Thus, understanding of the variation in gene expression profiles of cell populations and their effect on function are limited. To investigate the mononuclear cell composition in bovine milk, we analyzed a single-cell suspension from a milk sample. Additionally, we harvested cultured pbMECs to characterize gene expression in a homogeneous cell population. Using the Drop-seq technology, we generated single-cell RNA datasets of somatic milk cells and pbMECs. The final datasets after quality control filtering contained 7,119 and 10,549 cells, respectively. The pbMECs formed 14 indefinite clusters displaying intrapopulation heterogeneity, whereas the milk cells formed 14 more distinct clusters. Our datasets constitute a molecular cell atlas that provides a basis for future studies of milk cell composition and gene expression, and could serve as reference datasets for milk cell analysis.

Comparative Analysis of the Circular Transcriptome in Muscle, Liver, and Testis in Three Livestock Species.

Circular RNAs have been observed in a large number of species and tissues and are now recognized as a clear component of the transcriptome. Our study takes advantage of functional datasets produced within the FAANG consortium to investigate the pervasiveness of circular RNA transcription in farm animals. We describe here the circular transcriptional landscape in pig, sheep and bovine testicular, muscular and liver tissues using total 66 RNA-seq datasets. After an exhaustive detection of circular RNAs, we propose an annotation of exonic, intronic and sub-exonic circRNAs and comparative analyses of circRNA content to evaluate the variability between individuals, tissues and species. Despite technical bias due to the various origins of the datasets, we were able to characterize some features (i) (ruminant) liver contains more exonic circRNAs than muscle (ii) in testis, the number of exonic circRNAs seems associated with the sexual maturity of the animal. (iii) a particular class of circRNAs, sub-exonic circRNAs, are produced by a large variety of multi-exonic genes (protein-coding genes, long non-coding RNAs and pseudogenes) and mono-exonic genes (protein-coding genes from mitochondrial genome and small non-coding genes). Moreover, for multi-exonic genes there seems to be a relationship between the sub-exonic circRNAs transcription level and the linear transcription level. Finally, sub-exonic circRNAs produced by mono-exonic genes (mitochondrial protein-coding genes, ribozyme, and sno) exhibit a particular behavior. Caution has to be taken regarding the interpretation of the unannotated circRNA proportion in a given tissue/species: clusters of circRNAs without annotation were characterized in genomic regions with annotation and/or assembly problems of the respective animal genomes. This study highlights the importance of improving genome annotation to better consider candidate circRNAs and to better understand the circular transcriptome. Furthermore, it emphasizes the need for considering the relative "weight" of circRNAs/parent genes for comparative analyses of several circular transcriptomes. Although there are points of agreement in the circular transcriptome of the same tissue in two species, it will be not possible to do without the characterization of it in both species.

A 50-kb deletion disrupting the RSPO2 gene is associated with tetradysmelia in Holstein Friesian cattle.

BACKGROUND: Tetradysmelia is a rare genetic disorder that is characterized by an extremely severe reduction of all limb parts distal of the scapula and pelvic girdle. We studied a Holstein Friesian backcross family with 24 offspring, among which six calves displayed autosomal recessive tetradysmelia. In order to identify the genetic basis of the disorder, we genotyped three affected calves, five dams and nine unaffected siblings using a Bovine Illumina 50 k BeadChip and sequenced the whole genome of the sire. RESULTS: Pathological examination of four tetradysmelia cases revealed a uniform and severe dysmelia of all limbs. Applying a homozygosity mapping approach, we identified a homozygous region of 10.54 Mb on chromosome 14 (Bos taurus BTA14). Only calves that were diagnosed with tetradysmelia shared a distinct homozygous haplotype for this region. We sequenced the whole genome of the cases' sire and searched for heterozygous single nucleotide polymorphisms (SNPs) and small variants on BTA14 that were uniquely present in the sire and absent from 3102 control whole-genome sequences of the 1000 Bull Genomes Project, but none were identified in the 10.54-Mb candidate region on BTA14. Therefore, we subsequently performed a more comprehensive analysis by also considering structural variants and detected a 50-kb deletion in the targeted chromosomal region that was in the heterozygous state in the cases' sire. Using PCR, we confirmed that this detected deletion segregated perfectly within the family with tetradysmelia. The deletion spanned three exons of the bovine R-spondin 2 (RSPO2) gene, which encode three domains of the respective protein. R-spondin 2 is a secreted ligand of leucine-rich repeats containing G protein-coupled receptors that enhance Wnt signalling and is involved in a broad range of developmental processes during embryogenesis. CONCLUSIONS: We identified a 50-kb deletion on BTA14 that disrupts the coding sequence of the RSPO2 gene and is associated with bovine tetradysmelia. To our knowledge, this is the first reported candidate causal mutation for tetradysmelia in a large animal model. Since signalling pathways involved in limb development are conserved across species, the observed inherited defect may serve as a model to further elucidate fundamental pathways of limb development.

Beyond Back Splicing, a Still Poorly Explored World: Non-Canonical Circular RNAs.

Most of the circRNAs reported to date originate from back splicing of a pre-mRNA, and these exonic circRNAs are termed canonical circRNAs. Our objective was to provide an overview of all other (non-canonical) circRNAs that do not originate from the junction of two exons and to characterize their common properties. Those generated through a failure of intron lariat debranching are the best known, even though studies on them are rare. These circRNAs retain the 2'-5' bond derived from the intron lariat, and this feature probably explains the difficulties in obtaining efficient reverse transcription through the circular junction. Here, we provide an unprecedented overview of non-canonical circRNAs (lariat-derived intronic circRNAs, sub-exonic circRNAs, intron circles, tricRNAs), which all derive from non-coding sequences. As there are few data suggesting their involvement in cellular regulatory processes, we believe that it is early to propose a general function for circRNAs, even for lariat-derived circRNAs. We suggest that their small size and probably strong secondary structures could be major obstacles to their reliable detection. Nevertheless, we believe there are still several possible ways to advance our knowledge of this class of non-coding RNA.

In-Depth Analysis Reveals Production of Circular RNAs from Non-Coding Sequences.

The sequencing of total RNA depleted for ribosomal sequences remains the method of choice for the study of circRNAs. Our objective was to characterize non-canonical circRNAs, namely not originating from back splicing and circRNA produced by non-coding genes. To this end, we analyzed a dataset from porcine testis known to contain about 100 intron-derived circRNAs. Labelling reads containing a circular junction and originating from back splicing provided information on the very small contribution of long non-coding genes to the production of canonical circRNAs. Analyses of the other reads revealed two origins for non-canonical circRNAs: (1) Intronic sequences for lariat-derived intronic circRNAs and intron circles, (2) Mono-exonic genes (mostly non-coding) for either a new type of circRNA (including only part of the exon: sub-exonic circRNAs) or, even more rarely, mono-exonic canonical circRNAs. The most complex set of sub-exonic circRNAs was produced by RNase_MRP (ribozyme RNA). We specifically investigated the intronic circRNA of ATXN2L, which is probably an independently transcribed sisRNA (stable intronic sequence RNA). We may be witnessing the emergence of a new non-coding gene in the porcine genome. Our results are evidence that most non-canonical circRNAs originate from non-coding sequences.

Indication of Premelanosome Protein (PMEL) Expression Outside of Pigmented Bovine Skin Suggests Functions Beyond Eumelanogenesis.

The premelanosome protein (PMEL) is important for fibril formation within melanosomes during vertebrate melanogenesis. Fibrils form a matrix for pigment deposition within pigmented tissues such as skin and hair. PMEL mutations are known to modulate eumelanic pigmentation in vertebrates. However, in bovines, PMEL mutations were also found to alter pheomelanic pigmentation resulting in coat color dilution. Furthermore, epistatic effects of a mutated PMEL allele were detected in the phenotypic expression of the bovine hair defect "rat-tail syndrome" (RTS) characterized by charcoal coat color and hair deformation. Reports about PMEL gene expression in non-pigmented tissues raised the hypothesis that there may be unknown functions of the PMEL protein beyond eumelanin deposition to PMEL fibrils. In our study, we analysed the PMEL protein expression in pigmented skin and non-pigmented bovine tissues (non-pigmented skin, thyroid gland, rumen, liver, kidney, and adrenal gland cortex). We found that a processed form of the bovine PMEL protein is expressed in pigmented as well as in non-pigmented tissues, which is in line with gene expression data from targeted RT-PCR and whole transcriptome RNAseq analysis. The PMEL protein is located in membranes and within the cytosol of epithelial cells. Based on our data from bovine tissues, we concluded that at least in cattle PMEL potentially has additional, yet unexplored functions, which might contribute to effects of PMEL mutations on pheomelanin coat color dilution and charcoal coat color in RTS animals. However, indication of PMEL protein in unpigmented cells and tissues will require further confirmation in the future, because there have been no confirmed reports before, which had detected bovine PMEL protein with specific antibodies either in pigmented or unpigmented tissue.

Differentially Expressed miRNA-Gene Targets Related to Intramuscular Fat in Musculus Longissimus Dorsi of Charolais × Holstein F2-Crossbred Bulls.

Intramuscular fat (IMF) is a meat quality indicator associated with taste and juiciness. IMF deposition, influenced by genetic and non-genetic factors, occurs through a transcriptionally coordinated process of adipogenesis. MicroRNAs (miRNAs) are transcriptional regulators of vital biological processes, including lipid metabolism and adipogenesis. However, in bovines, limited data on miRNA profiling and association with divergent intramuscular fat content, regulated exclusively by genetic parameters, have been reported. Here, a microarray experiment was performed to identify and characterize the miRNA expression pattern in the Musculus longissimus dorsi of F2-cross (Charolais × German Holstein) bulls with high and low IMF. A total of 38 differentially expressed miRNAs (DE miRNAs), including 33 upregulated and 5 downregulated (corrected p-value ≤ 0.05, FC ≥ ±1.2), were reported. Among DE miRNAs, the upregulated miRNAs miR-105a/b, miR-695, miR-1193, miR-1284, miR-1287-5p, miR-3128, miR-3178, miR-3910, miR-4443, miR-4445 and miR-4745, and the downregulated miRNAs miR-877-5p, miR-4487 and miR-4706 were identified as novel fat deposition regulators. DE miRNAs were further analyzed, along with previously identified differentially expressed genes (DEGs) from the same samples and predicted target genes, using multiple bioinformatic approaches, including target prediction tools and co-expression networks, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. We identified DE miRNAs and their gene targets associated with bovine intramuscular adipogenesis, and we provide a basis for further functional investigations.