The use of SNP markers for cattle breed identification.

A potential application of single nucleotide polymorphisms (SNPs) in animal husbandry and production is identification of the animal breed. In this study, using chosen marker selection methods and genotypic data obtained with the use of Illumina Bovine SNP50 BeadChip for individuals belonging to ten cattle breeds, the reduced panels containing the most informative SNP markers were developed. The suitability of selected SNP panels for the effective and reliable assignment of the studied individuals to the breed of origin was checked by three allocation algorithms implemented in GeneClass 2. The studied breeds set included both Polish-native breeds under the genetic resources conservation programs and highly productive breeds with a global range. For all of the tested marker selection methods ("delta" and two FST-based variants), two separate methodological approaches of marker assortment were used and three marker panels were created with 96, 192, and 288 SNPs respectively, to determine the minimum number of markers required for effective differentiation of the studied breeds. Moreover, the usefulness of the most effective panels of markers to assess the population structure and genetic diversity of the analyzed breeds was examined. The conducted analyses showed the possibility of using SNP subsets from medium-density genotypic microarrays to distinguish breeds of cattle kept in Poland and to analyze their genetic structure.

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Bioinformatic analysis of endometrial miRNA expression profile at day 26-28 of pregnancy in the mare.

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26-28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26-28) and endometrium during the mid-luteal phase of the estrous cycle (day 10-12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26-28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

Standardization of the protocol for oral cavity examination and collecting of the biological samples for microbiome research using the next-generation sequencing (NGS): own experience with the COVID-19 patients.

To date, publications have shown that compositions of oral microbiota differ depending on their habitats (e.g. tongue, tonsils, pharynx). The absence of set standards for the choice of the areas and conditions of material collection makes the oral microbiome one of the most difficult environments for a comparative analysis with other researchers, which is a meaningful limitation during an assessment of the potential effects of microorganisms as biomarkers in the courses of various human diseases. Therefore, standardisation of basic conditions of a dental examination and collection of material for the next generation sequencing (NGS) is worth attempting. The standardisation of the dental exam and collection of the clinical materials: saliva, swab from the tongue ridge, hard palate, palatine tonsils and oropharynx, supragingival plaque and subgingival plaque. Protocol involved the patients (n = 60), assigned to 3 groups: I-COVID-19 convalescents who received antibiotics, n = 17, II-COVID-19 convalescents, n = 23 and III-healthy individuals, n = 20. The collected biological samples were used to conduct NGS (16S rRNA). The conditions of patient preparation for collecting biological materials as well as the schedule of dental examination, were proposed. Based on the research conducted, we have indicated the dental indicators that best differentiate the group of COVID-19 patients (groups I and II) from healthy people (group III). These include the DMFT, D and BOP indices. The use of alpha and beta diversity analysis provided an overall insight into the diversity of microbial communities between specific niches and patient groups. The most different diversity between the studied group of patients (group II) and healthy people (group III) was noted in relation to the supragingival plaque. The order of activities during the dental exam as well as while collecting and securing clinical materials is particularly important to avoid technical errors and material contamination which may result in erroneous conclusions from the analyses of the results of sensitive tests such as the NGS. It has been shown that the dental indices: DMFT, D number, PI and BOP are the best prognostic parameters to assess the oral health. Based on beta diversity the most sensitive niche and susceptible to changes in the composition of the microbiota is the supragingival plaque. The procedures developed by our team can be applied as ready-to-use forms in studies conducted by other researchers.

Oral microbiota study of the patients after hospitalisation for COVID-19, considering selected dental indices and antibiotic therapy using the next generation sequencing method (NGS).

Background: Poor oral hygiene and the increased incidence and severity of periodontitis may exacerbate SARS-CoV-2 infection. The aim was to evaluate the oral microbiota of 60 participants divided into groups: COVID-19 convalescents who received antibiotics during hospitalization (I), COVID-19 convalescents without antibiotic therapy (II) and healthy individuals (III). Materials and Methods: Dental examination was conducted, and oral health status was evaluated using selected dental indexes. Clinical samples (saliva, dorsal swabs, supragingival and subgingival plaque) were collected and used for metagenomic library to the next-generation sequencing (NGS) preparation. Results: Each of the clinical materials in particular groups of patients showed a statistically significant and quantitatively different bacterial composition. Patients from group I showed significantly worse oral health, reflected by higher average values of dental indexes and also a higher percentage of Veillonella, Tannerella, Capnocytophaga and Selenomonas genera in comparison to other groups. Additionally, a statistically significant decrease in the amount of Akkermansia type in both groups with COVID-19 was observed for all materials. Conclusions: The primary factor affecting the composition of oral microbiota was not the SARS-CoV-2 infection itself, but the use of antibiotic therapy. The increased percentage of pro-inflammatory pathogens observed in COVID-19 patients underscores the importance of preventing periodontal disease and improving oral hygiene in the future.

The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis.

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-ß1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-ß1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA).

Transcriptome and methylome sequencing reveals altered long non-coding RNA genes expression and their aberrant DNA methylation in equine sarcoids.

Recent publications confirmed that long non-coding RNAs (lncRNAs) perform an essential function in gene-specific transcription regulation. Nevertheless, despite its important role, lncRNA has not yet been described in equine sarcoids, the skin neoplasia of horses. Therefore, the aim of this study is to deepen the knowledge about lncRNA expression in the pathogenesis of equine sarcoids and provide new insight into the regulatory function of lncRNA in the bovine papillomavirus-dependent neoplasia of horse dermal tissues. RNA sequencing (RNA-seq) data from 12 equine sarcoid samples and the corresponding controls were reanalyzed in this study. A total of 3396 differentially expressed (DE) lncRNAs and 128 DElncRNA-DE genes (DEGs) pairs were identified. Differentially expressed lncRNAs predicted target genes were enriched in pathways associated with inter alia the extracellular matrix disassembly and cancer pathways. Furthermore, methylation data from the same samples were integrated into the analysis, and 12 DElncRNAs were described as potentially disturbed by aberrant methylation. In conclusion, this study presents novel data about lncRNA's role in the pathogenesis of equine sarcoids.

Application of Nanopore Sequencing for High Throughput Genotyping in Horses.

Nanopore sequencing is a third-generation biopolymer sequencing technique that relies on monitoring the changes in an electrical current that occur as nucleic acids are passed through a protein nanopore. Increasing quality of reads generated by nanopore sequencing systems encourages their application in genome-wide polymorphism detection and genotyping. In this study, we employed nanopore sequencing to identify genome-wide polymorphisms in the horse genome. To reduce the size and complexity of genome fragments for sequencing in a simple and cost-efficient manner, we amplified random DNA fragments using a modified DOP-PCR and sequenced the resulting products using the MinION system. After initial filtering, this generated 28,426 polymorphisms, which were validated at a 3% error rate. Upon further filtering for polymorphism and reproducibility, we identified 9495 SNPs that reflected the horse population structure. To conclude, the use of nanopore sequencing, in conjunction with a genome enrichment step, is a promising tool that can be practical in a variety of applications, including genotyping, population genomics, association studies, linkage mapping, and potentially genomic selection.

Methylome and transcriptome data integration reveals aberrantly regulated genes in equine sarcoids.

DNA methylation is a key mechanism in transcription regulation, and aberrant methylation is a common and important mechanism in tumor initiation, maintenance, and progression. To find genes that are aberrantly regulated by altered methylation in horse sarcoids, we used reduced representation bisulfite sequencing (RRBS) accompanied by RNA sequencing (RNA-Seq) for methylome (whole genome DNA methylation sequencing) and transcriptome profiling, respectively. We found that the DNA methylation level was generally lower in lesion samples than in controls. In the analyzed samples, a total of 14,692 differentially methylated sites (DMSs) in the context of CpG (where cytosine and guanine are separated by a phosphate), and 11,712 differentially expressed genes (DEGs) were identified. The integration of the methylome and transcriptome data suggests that aberrant DNA methylation may be involved in the deregulation of expression of the 493 genes in equine sarcoid. Furthermore, enrichment analysis of the genes demonstrated the activation of multiple molecular pathways related to extracellular matrix (ECM), oxidative phosphorylation (OXPHOS), immune response, and disease processes that can be related to tumor progression. The results provide further insight into the epigenetic alterations in equine sarcoids and provide a valuable resource for follow-up studies to identify biomarkers for predicting susceptibility to this common condition in horses.

Seeds of Stevia rebaudiana Bertoni as a Source of Plant Growth-Promoting Endophytic Bacteria with the Potential to Synthesize Rebaudioside A.

In this study, a new strain of Pantoea vagans, SRS89, was isolated from surface-sterilized stevia seeds. The isolate was evaluated using morphological, molecular, and biochemical methods. The bacterium was 1.5 µm long, yellowish in color, and classified as Gram-negative. Whole genome sequencing of our strain revealed the presence of a 4,610,019 bp chromosome, and genome annotation resulted in the detection of 4283 genes encoding 4204 putative coding sequences. Phylogenic analysis classified the genome of our strain close to the MP7 and LMG 24199 strains of P. vagans. Functional analysis showed that the highest number of genes within the analyzed bacterium genome were involved in transcription, amino acid transport and metabolism, and carbohydrate transport and metabolism. We also identified genes for enzymes involved in the biosynthesis of carotenoids and terpenoids. Furthermore, we showed the presence of growth regulators, with the highest amount noted for gibberellic acid A3, indole-3-acetic acid, and benzoic acid. However, the most promising property of this strain is its ability to synthesize rebaudioside A; the estimated amount quantified using reversed-phase (RP)-HPLC was 4.39 mg/g of the dry weight of the bacteria culture. The isolated endophytic bacterium may be an interesting new approach to the production of this valuable metabolite.

Assessment and Distribution of Runs of Homozygosity in Horse Breeds Representing Different Utility Types.

The present study reports runs of homozygosity (ROH) distribution in the genomes of six horse breeds (571 horses in total) representing three horse types (primitive, light, and draft horses) based on the 65k Equine BeadChip assay. Of major interest was the length, quantity, and frequency of ROH characteristics, as well as differences between horse breeds and types. Noticeable differences in the number, length and distribution of ROH between breeds were observed, as well as in genomic inbreeding coefficients. We also identified regions of the genome characterized by high ROH coverage, known as ROH islands, which may be signals of recent selection events. Eight to fourteen ROH islands were identified per breed, which spanned multiple genes. Many were involved in important horse breed characteristics, including WFIKNN2, CACNA1G, STXBP4, NOG, FAM184B, QDPR, LCORL, and the zinc finger protein family. Regions of the genome with zero ROH occurrences were also of major interest in specific populations. Depending on the breed, we detected between 2 to 57 no-ROH regions and identified 27 genes in these regions that were common for five breeds. These genes were involved in, e.g., muscle contractility (CACNA1A) and muscle development (miR-23, miR-24, miR-27). To sum up, the obtained results can be furthered analyzed in the topic of identification of markers unique for specific horse breed characteristics.

Endophytic bacteria from in vitro culture of Leucojum aestivum L. a new source of galanthamine and elicitor of alkaloid biosynthesis.

Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L. aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P. lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A1, abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P. lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis.

Another lesson from unmapped reads: in-depth analysis of RNA-Seq reads from various horse tissues.

In recent years, a vast amount of sequencing data has been generated and large improvements have been made to reference genome sequences. Despite these advances, significant portions of reads still do not map to reference genomes and these reads have been considered as junk or artificial sequences. Recent studies have shown that these reads can be useful, e.g., for refining reference genomes or detecting contaminating microorganisms present in the analyzed biological samples. A special case of this is RNA sequencing (RNA-Seq) reads that come from tissue transcriptomes. Unmapped reads from RNA-Seq have received much less attention than those from whole-genome sequencing. In particular, in the horse, an analysis of unmapped RNA reads has not been performed yet. Thus, in this study, we analyzed the unmapped reads originating from the RNA-Seq performed through the Functional Annotation of Animal Genomes (FAANG) project in the horse, using eight different tissues from two mares. We demonstrated that unmapped reads from RNA-Seq could be easily assembled into transcripts relating to many important genes present in the sequences of other mammals. Large portions of these transcripts did not have coding potential and, thus, can be considered as non-coding RNA. Moreover, reads that were not mapped to the reference genome but aligned to the entries in NCBI database of horse proteins were enriched for biological processes that largely correspond to the functions of organ from which RNA was isolated and thus are presumably true transcripts of genes associated with cell metabolism in those tissues. In addition, a portion of reads aligned to the common pathogenic or neutral microbiota, of which the most common was Brucella spp. These data suggest that unmapped reads can be an important target for in-depth analysis that may substantially enrich results of initial RNA-Seq experiments for various tissues and organs.

MicroRNA gene methylation landscape in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Aberrant DNA methylation is an important mechanism by which the normal patterns of microRNA expression are disrupted in human cancers including B-cell precursor acute lymphoblastic leukemia (BCP ALL), the most common pediatric malignancy. OBJECTIVES: To characterize the methylation profile landscape of microRNA genes in BCP ALL patients. MATERIAL AND METHODS: We employed Infinium® MethylationEPIC BeadChip Arrays to measure the methylation of microRNA genes from bone marrow samples of children with BCP ALL (n = 38) and controls without neoplasms (n = 4). RESULTS: This analysis revealed differential methylation of the microRNA genes in the pediatric BCP ALL when compared to the control. A subcluster amongst BCP ALL patients with TCF3-PBX1 genetic subtype was also observed. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-related phenotypes are enriched by the genes with hyperand hypomethylated sites located in promoters as well as gene bodies. The top 3 miRNA genes, promoters of which were the most statistically significantly hypermethylated in BCP ALL were MIR1273G, MIR1304 and MIR663, and the top 3 hypomethylated were MIR4442, MIR155 and MIR3909. CONCLUSIONS: In this study, a different microRNA genes methylation landscape was shown in pediatric BCP ALL compared to children without neoplasms. A visible subcluster among BCP ALL samples consisted of individuals with TCF3-PBX1 genetic subtype. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-connected phenotypes were found, associated with genes with hyperand hypomethylated sites located on promoters as well as gene bodies.

Pet ownership in pregnancy and methylation pattern in cord blood.

Having pets in the house during the first years of life has been shown to protect against allergies. However, the result of different studies is heterogeneous. The aim of this study was to evaluate the methylation pattern in cord blood in relation to pet ownership during pregnancy.We investigated the methylation patterns of 96 cord blood samples, participants of the Epigenetic Hallmark of Maternal Atopy and Diet-ELMA project, born to mothers who either owned pets (n = 32) or did not own pets (n = 64) during their pregnancy. DNA from cord blood was analysed using the Infinium methylation EPIC. For statistical analysis, RnBeads software was applied.We found 113 differentially methylated sites (DMs) in the covariate-adjusted analysis (FDR p < 0.05), with small methylation differences. The top DMs were associated with genes: UBA7, THRAP3, GTDC1, PDE8A and SBK2. In the regional analysis, two promoter regions presented with significance: RN7SL621P and RNU6-211P. Cis-regulatory element analysis revealed significant associations with several immune-related pathways, such as regulation of IL18, Toll signalling, IL6 and complement.We conclude that pet exposure during pregnancy causes subtle but significant changes in methylation patterns in cord blood, which are reflected in the biological processes governing both innate and adaptive immune responses.

Peroxisome Proliferator-Activated Receptor γ, but Not α or G-Protein Coupled Estrogen Receptor Drives Functioning of Postnatal Boar Testis-Next Generation Sequencing Analysis.

Porcine tissue gene expression is highly similar to the expression of homologous genes in humans. Based on this fact, the studies on porcine tissues can be employed to understand human physiology and to predict or treat diseases. Our prior studies clearly showed that there was a regulatory partnership of the peroxisome proliferator-activated receptor (PPAR) and the G-protein coupled membrane estrogen receptor (GPER) that relied upon the tumorigenesis of human and mouse testicular interstitial cells, as well as the PPAR-estrogen related receptor and GPER-xenoestrogen relationships which affected the functional status of immature boar testes. The main objective of this study was to identify the biological processes and signaling pathways governed by PPARα, PPARγ and GPER in the immature testes of seven-day-old boars after pharmacological receptor ligand treatment. Boar testicular tissues were cultured in an organotypic system with the respective PPARα, PPARγ or GPER antagonists. To evaluate the effect of the individual receptor deprivation in testicular tissue on global gene expression, Next Generation Sequencing was performed. Bioinformatic analysis revealed 382 transcripts with altered expression. While tissues treated with PPARα or GPER antagonists showed little significance in the enrichment analysis, the antagonists challenged with the PPARγ antagonist displayed significant alterations in biological processes such as: drug metabolism, adhesion and tubule development. Diverse disruption in the Notch signaling pathway was also observed. The findings of our study proposed that neither PPARα nor GPER, but PPARγ alone seemed to be the main player in the regulation of boar testes functioning during early the postnatal developmental window.

Transcriptome analysis of human Leydig cell tumours reveals potential mechanisms underlying its development.

Leydig cell tumours are the most common sex cord-stromal tumours. In the last years, apparent increased incidence is noted while aetiology of the tumour is still unknown. Therefore, here, we focused on the genetics of Leydig cell tumours using the next-generation sequencing. Leydig cell micronodules were revealed in patients with azoospermia who were qualified for testicular biopsy. Complete gene set of Leydig cell tumours was compared with transcriptome of healthy Leydig cells obtained from donors. Bioinformatic analysis of the obtained sequencing data revealed alterations in expression of 219 transcripts. We showed, for the first time, that a significant proportion of differentially expressed genes is directly involved in regulation of apoptotic process, which downregulation might be important to Leydig cell tumour development. Additionally, we found a significant upregulation of heat shock protein genes that might be a unique feature of Leydig cell tumours when compared to other tumour types. Our study offers fundamental transcriptomic data for future studies on human Leydig cell tumour that are crucial to determine its causes. Moreover, presented here the in-depth analysis and discussion of alterations observed in tumour transcriptome may be important for the diagnosis and therapy of this pathology.

Single Nucleotide Polymorphism Discovery and Genetic Differentiation Analysis of Geese Bred in Poland, Using Genotyping-by-Sequencing (GBS).

Poland is the largest European producer of goose, while goose breeding has become an essential and still increasing branch of the poultry industry. The most frequently bred goose is the White Koluda® breed, constituting 95% of the country's population, whereas geese of regional varieties are bred in smaller, conservation flocks. However, a goose's genetic diversity is inaccurately explored, mainly because the advantages of the most commonly used tools are strongly limited in non-model organisms. One of the most accurate used markers for population genetics is single nucleotide polymorphisms (SNP). A highly efficient strategy for genome-wide SNP detection is genotyping-by-sequencing (GBS), which has been already widely applied in many organisms. This study attempts to use GBS in 12 conservative goose breeds and the White Koluda® breed maintained in Poland. The GBS method allowed for the detection of 3833 common raw SNPs. Nevertheless, after filtering for read depth and alleles characters, we obtained the final markers panel used for a differentiation analysis that comprised 791 SNPs. These variants were located within 11 different genes, and one of the most diversified variants was associated with the EDAR gene, which is especially interesting as it participates in the plumage development, which plays a crucial role in goose breeding.

Application of the targeted sequencing approach reveals the single nucleotide polymorphism (SNP) repertoire in microRNA genes in the pig genome.

MicroRNAs (miRNAs) are recognized as gene expression regulators, indirectly orchestrating a plethora of biological processes. Single nucleotide polymorphism (SNP), one of the most common genetic variations in the genome, is established to affect miRNA functioning and influence complex traits and diseases. SNPs in miRNAs have also been associated with important production traits in livestock. Thus, the aim of our study was to reveal the SNP variability of miRNA genes in the genome of the pig, which is a significant farm animal and large-mammal human model. To this end, we applied the targeted sequencing approach, enabling deep sequencing of specified genomic regions. As a result, 73 SNPs localized in 50 distinct pre-miRNAs were identified. In silico analysis revealed that many of the identified SNPs influenced the structure and energy of the hairpin precursors. Moreover, SNPs localized in the seed regions were shown to alter targeted genes and, as a result, enrich different biological pathways. The obtained results corroborate a significant impact of SNPs on the miRNA processing and broaden the state of knowledge in the field of animal genomics. We also report the targeted sequencing approach to be a promising alternative for the whole genome sequencing in miRNA genes focused studies.

Suitability of Pedigree Information and Genomic Methods for Analyzing Inbreeding of Polish Cold-Blooded Horses Covered by Conservation Programs.

Traditionally, pedigree-based relationship coefficients were used to manage inbreeding and control inbreeding depression that occurs within populations. The extensive incorporation of genomic data in livestock breeding creates the opportunity to develop and implement methods to manage populations at the genomic level. Consequently, the realized proportion of the genome that two individuals share can be more accurately estimated instead of using pedigree information to estimate the expected proportion of shared alleles. To make use of this improvement, in this study we evaluated the genomic inbreeding measures in the Polish conserved cold-blooded horse population and compared the data with the traditional measures of inbreeding. Additionally, an ancestry fractions/proportions from Admixture software were tested as an estimate of lineage (ancestry coefficient) used for horses qualifying for the conservation program. The highest correlation of pedigree-based (FPED) and genomic inbreeding estimates was found for FROH (runs of homozygosity-based F coefficient) and FUNI (F coefficient based on the correlation between uniting gametes). FROH correlation with FPED tended to increase as the number of generations registered as pedigree increased. While lineage and gene contributions (Q) from Admixture software correlated, they showed poor direct compliance; hence, Q-value cannot be recommended as the estimate of pedigree-based lineage. All these findings suggest that the methods of genomics should be considered as an alternative or support in the analysis of population structure in conservative breeding that can help control inbreeding in rare horse populations.