Genotyping-by-sequencing performance in selected livestock species.

Application of next generation sequencing for large scale genotyping in livestock is limited by high costs and challenging data analysis process. However, available restriction enzyme-based enrichment techniques like e.g. genotyping-by-sequencing (GBS) are promising tools allowing reduction of financial outlies by a high sample multiplexing and narrowing down the sequenced genome areas to the randomly distributed read tags. In this study, we tested the performance of standard, PstI endonuclease-adapted GBS protocol for population genetics in cattle, horse and sheep with application of different, including low-depth sequencing setups. It was found that the detected SNPs display desirable polymorphism parameters and are evenly scattered across the whole genome including gene coding regions. It was also shown that the SNPs can be successfully applied in population genetics, revealing the genetic differentiation of the studied breeds. The GBS approach represents a cost-effective alternative to existing genotyping methods which may find adoption in various research applications.

Monocyte subpopulations display disease-specific miRNA signatures depending on the subform of Spondyloarthropathy.

Spondyloarthropathies (SpA) are a family of rheumatic disorders that could be divided into axial (axSpA) and peripheral (perSpA) sub-forms depending on the disease clinical presentation. The chronic inflammation is believed to be driven by innate immune cells such as monocytes, rather than self-reactive cells of adaptive immune system. The aim of the study was to investigate the micro-RNA (miRNA) profiles in monocyte subpopulations (classical, intermediate and non-classical subpopulations) acquired from SpA patients or healthy individuals in search for prospective disease specific and/or disease subtype differentiating miRNA markers. Several SpA-specific and axSpA/perSpA differentiating miRNAs have been identified that appear to be characteristic for specific monocyte subpopulation. For classical monocytes, upregulation of miR-567 and miR-943 was found to be SpA-specific, whereas downregulation of miR-1262 could serve as axSpA-differentiating, and the expression pattern of miR-23a, miR-34c, mi-591 and miR-630 as perSpA-differentiating markers. For intermediate monocytes, expression levels of miR-103, miR-125b, miR-140, miR-374, miR-376c and miR-1249 could be used to distinguish SpA patients from healthy donors, whereas the expression pattern of miR-155 was identified as characteristic for perSpA. For non-classical monocytes, differential expression of miR-195 was recognized as general SpA indicator, while upregulation of miR-454 and miR-487b could serve as axSpA-differentiating, and miR-1291 as perSpA-differentiating markers. Our data indicate for the first time that in different SpA subtypes, monocyte subpopulations bear disease-specific miRNA signatures that could be relevant for SpA diagnosis/differentiation process and may help to understand SpA etiopathology in the context of already known functions of monocyte subpopulations.

Characterization of PRNP and SPRN coding regions from atypical scrapie cases diagnosed in Poland.

Scrapie, a fatal transmissible spongiform encephalopathy (TSE) occurs in two phenotypes: classical and atypical. Many authors point out that the polymorphism of three codons (136, 154, 171) of the PRNP (PrP gene) is associated with a sheep susceptibility to classical scrapie. Until now, only one PRNP gene variant coding phenylalanine at codon 141 has been found to be associated with atypical scrapie. Another recently identified and interesting candidate gene for scrapie susceptibility in sheep is an SPRN gene coding for Shadoo protein (Sho). Sho is a highly interspecies conserved protein and an insertion/deletion (indel) found in a sheep Sho gene was associated with classical scrapie occurrence. Here we determined the polymorphism of PRNP and SPRN genes in nine atypical scrapie cases (six in native born sheep and three in imported sheep) and compared these results with a control group of healthy animals comprising six corresponding Polish sheep breeds. In atypical scrapie cases five PRNP diplotypes were identified: A(136)R(154)Q(171)/ARQ, AHQ/ARQ, ARR/ARQ, ARR/AHQ and AHQ/AHQ. The ARR/AHQ diplotype was found only in imported sheep. A previously unobserved SNP in PRNP (E224K) was also found in both atypical scrapie and in a few control animals. In the ORF of the SPRN gene, six SNPs and one indel were identified. None of these variations was exclusive for scrapie animals and they were probably, naturally occurring polymorphisms. Special attention was given to the 6-bp indel SPRN polymorphism which was previously associated with classical scrapie occurrence.

Variability of mRNA abundance of leukemia inhibitory factor gene (LIF) in porcine ovary, oviduct and uterus tissues.

The leukemia inhibitory factor (LIF) gene encodes a pleiotropic cytokine which is produced by the endometrium and plays an important role in implantation and early embryonic development. Because of its function, LIF gene is considered as a candidate gene for litter size in many mammalian species including pig. The aim of present study was to evaluate the expression of LIF gene in the porcine ovary, oviduct and two regions of uterus (corpus uteri, cornu uteri) in prepubertal and pubertal gilts. In order to precise estimation of LIF transcript abundance we evaluated the stability of expression for several candidate housekeeping genes in investigated tissues across different breeds and different stage of oestrus cycle. The geNorm analysis indicated that the most stable reference genes across analyzed tissues were: OAZ1 and RPL27. The analysis conducted separately for each tissue confirmed that the most stable gene was OAZ1 in all tissues expect oviduct (the most stable was RPL27 gene). In prepubertal pigs, the highest level of the LIF expression was obtained in both regions of uterus compare to ovary and oviduct tissues (P < 0.01). A similar trend in LIF expression pattern was observed in follicular phase-the significantly highest transcript level was obtained in cornu uteri, it was about ninefold higher than in ovary (P < 0.05). In luteal stage the highest expression was in corpus uteri. In pig, the high expression in luteal phases suggested that, LIF may be mainly secreted in respond to the increased of progesterone concentration and it can be connected with the preparation of the uterus for implantation.

Current Analytical Methods and Research Trends Are Used to Identify Domestic Pig and Wild Boar DNA in Meat and Meat Products.

The pig, one of the most important livestock species, is a meaningful source of global meat production. It is necessary, however, to prove whether a food product that a discerning customer selects in a store is actually made from pork or venison, or does not contain it at all. The problem of food authenticity is widespread worldwide, and cases of meat adulteration have accelerated the development of food and the identification methods of feed species. It is worth noting that several different molecular biology techniques can identify a porcine component. However, the precise differentiation between wild boar and a domestic pig in meat products is still challenging. This paper presents the current state of knowledge concerning the species identification of the domestic pig and wild boar DNA in meat and its products.

Genetic Variability and Population Structure of Polish Konik Horse Maternal Lines Based on Microsatellite Markers.

The aim of the conservation programme is to maintain the population size of endangered livestock breeds of less economic importance at a level that ensures the survival of the breed, the preservation of genetic diversity, and the preservation of as many pedigree lines as possible. The Polish Konik, a native Polish primitive-type horse breed and is one of the breeds included in such a programme in Poland. Presently, there are only 16 (of the 35 maternal lines known in 1962), some of which are endangered. We examined the genetic variability and structure of the Polish Konik maternal lines (176 individuals) on the basis of the pedigree data and 17 microsatellite markers (STRs) from parentage testing. The overall mean number of alleles was 7.647 (±0.411), the effective number of alleles was 3.935 (±0.271), the mean number of alleles for which the frequency was equal to or lower than 5% was 4.471 (±0.286), and the mean information index was 1.506 (±0.087). The structure of the population and admixture patterns were calculated with the Structure and Structure Harvester software. The structural analysis indicated three likely genetic clusters; as the most optimal K value was estimated as 3, with ∆K of 15.4188. The F-statistics results indicated a low level of inbreeding (average inbreeding coefficient FIT was 0.0188, coefficient of differentiation FST was 0.0304, and mean inbreeding index value FIS was -0.0119). Variability monitoring should be carried out in order to avoid inbreeding depression, while breeding strategies should be designed to prevent the decrease of genetic variability in the Polish horse breed and to sustain the active female lines.

Common Myelin Regulatory Factor Gene Variants Predisposing to Excellence in Sports.

In all sport disciplines, excellent coordination of movements is crucial for achieving mastery. The ability to learn new motor skills quickly and effectively is dependent on efficient myelination which varies between individuals. It has been suggested that these differences may play a role in athletic performance. The process of myelination is under transcriptional control by Myelin Regulatory Factor (MYRF) as well as other transcription factors (SOX10 and OLIG2). We analyze a panel of 28 single nucleotide polymorphisms (SNPs) located within the frequencies of common variants of MYRF, SOX10 and OLIG2 genes in professional athletes compared to non-athletes. No significant differences were detected after correction for multiple testing by false discovery rate (FDR) for any of the models tested. However, some deviations from the expected distribution was found for seven SNPs (rs174528, rs139884, rs149435516 and rs2238001, rs7943728, rs61747222, and rs198459). The MYRF alleles rs7943728 and rs61747222 showed a correlation with the level of sport achievement among the athletes. Even though the athletes did not differ from the non-athlete controls in the distribution of most SNPs analyzed, some interesting differences of several variants were noted. Presented results indicate that genetic variants of MYRF and SOX10 could be genetic factors weakly predisposing for successful athletic performance.

Genetic Diversity and Population Structure of Polish Konik Horse Based on Individuals from All the Male Founder Lines and Microsatellite Markers.

The Polish Konik horse is a primitive native breed included in the genetic resource conservation program in Poland. After World War II, intensive breeding work began, aimed at rebuilding this breed. Now, the whole Polish Konik population is represented by six male founder lines (Wicek, Myszak, Glejt I, Goraj, Chochlik and Liliput). Individuals representing all six paternal lineages were selected based on their breeding documentation. We performed a fragment analysis with 17 microsatellite markers (STRs) recommended by the International Society for Animal Genetics (ISAG). The genetic diversity and structure within the paternal lineages and the whole of the studied group were investigated. The average allelic richness was 6.497 for the whole studied group. The fixation index (FST; measure of population differentiation) was low (about 3%), the mean inbreeding coefficient (FIT) was low and close to 0, and the mean inbreeding index value (FIS) was negative. The mean expected heterozygosity was established at 0.7046 and was lower than the observed heterozygosity. The power of discrimination and power of exclusion were 99.9999%. The cumulative parentage exclusion probability equaled 99.9269% when one parental genotype was known and 99.9996% with both parents' genotypic information was available. About 3% of the genetic variation was caused by differences in the breed origin and about 97% was attributed to differences among individuals. Our analysis revealed that there has been no inbreeding in the Polish Konik breed for the studied population. The genetic diversity was high, and its parameters were similar to those calculated for native breeds from other countries reported in the literature. However, due to the small number of breed founders and paternal lineages with unknown representation, the population's genetic diversity and structure should be monitored regularly.

Frequency of genotypes in the PrP prion protein gene locus in the Polish sheep population.

Scrapie is an invariably fatal transmissible neurodegenerative disease of sheep, goats and moufflons, characterised by ataxia, lower body weight and changes in behaviour. The aim of this study was to determine the genotype distribution at codons 136, 154 and 171 of the PrP locus in sheep bred in Poland. The genotypes of 801 sheep representing 10 different breeds and crossbreds were analysed using real-time PCR allele discrimination method. The combination of point mutations of the three codons (136, 154 and 171) created twelve different genotypes in the PrP locus classified into five categories of risk. The highest frequency for the ARR/ARR genotype was characteristic of the Pogórze and Berrichon du Cher breeds (72.22% and 63:25%, respectively). In the Romanov breed (1.52%) and in crossbred sheep (2.61%) the VRQ/VRQ genotype was observed.

Impact of Bovine Lipocalin-2 Gene on the Antioxidant Activity of Milk from Polish Holstein-Friesian Cows.

In the recent years, antioxidant properties of food products have become an important aspect for consumers. Milk is a very good source of easily absorbable proteins and minerals, as well as a valuable source of antioxidants. Lipocalin-2 (LCN2), given that, inter alia, it is produced in large quantities by various types of cells in response to oxidative stress caused by physical or chemical factors, it can be considered a protein that determines the total antioxidant capacity of milk. The main objective of this study was to analyze polymorphisms within the lipocalin-2 gene and to determine their impact on antioxidant activity of milk from Holstein-Friesian cows. The genotyping was carried out by sequencing of PCR products. To determine the antioxidant activity of milk, the Trolox equivalent antioxidant capacity (TEAC) method was used. A total of four polymorphic sites were identified in the examined segment of the bovine lipocalin-2 gene. It was shown that cows of the CC genotype at the locus g.98793763G>C produced milk of significantly higher antioxidant capacity. The antioxidant capacity of milk also varied according to the age of cows, their daily milk yield, and SCC in milk.

Polymorphism of bovine lipocalin-2 gene and its impact on milk production traits and mastitis in Holstein Friesian cattle

Background: Mastitis is one of the most serious diseases of dairy cattle, causing substantial financial losses. While predisposition to reduced somatic cell count in milk has been considered for in cattle breeding programs as the key indicator of udder health status, scientists are seeking genetic markers of innate immune response, which could be helpful in selecting cows with improved immunity to mastitis. Lipocalin-2 (LCN2) is a protein involved in the response of the immune system by eliminating iron ions which are necessary for the growth of pathogenic bacteria, so LCN2 may be considered as a natural bacteriostatic agent and could become a marker of infection. Results: A total of five SNPs were identified in LCN2 gene (one in the promoter, three in exon 1, and one in intron 1). A single haplotype block was identified. The locus g.98793763GNC was found to have a significant impact on protein levels in milk, and alleles of this locus were identified to have a significant positive dominance effect on this trait. None of the four analysed loci had a statistically significant impact on the milk yield, fat levels in milk or the somatic cell score. LCN-2 gene had no significant impact on the incidence of mastitis in the cows. Conclusions: Although the identified SNPs were not found to have any impact on the somatic cell count or the incidence of mastitis in cows, it seems that further research is necessary, covering a larger population of cattle, to confirm the association between lipocalin-2 and milk production traits and mastitis.

Genetic polymorphism of Hucul horse population based on 17 microsatellite loci.

Short tandem repeat (STR) loci, i.e. microsatellites are a class of genetic markers commonly used for population studies and parentage control. This study determined the usefulness of microsatellite markers recommended by International Society for Animal Genetics (ISAG) for identification and pedigree analysis in horses based on the example of Polish Hucul horse population (Equus caballus). The set of seventeen microsatellites loci was tested (AHT4, AHT5, ASB2, HMS2, HMS3, HMS6, HMS7, HTG10, HTG4, HTG6, HTG7, VHL20, ASB17, ASB23, CA425, HMS1, LEX3) for 216 individuals. All samples were genotyped and mean number of alleles per locus was estimated (7.00). Means of observed (Ho) and expected (He) heterozygosity were calculated 0.7288 and 0.7027, respectively. The observed heterozygosity was similar to the results of research on Hucul horse population in another area of Carpathians Mountains. The average polymorphism information content (PIC) for analyses of seventeen microsatellite markers indicates the usefulness of this set of markers for Hucul horse parentage testing.

Expression and imprinting analysis of the NESP55 gene in pigs.

Most imprinted genes play important roles in a mammalian development. One of them is GNAS complex locus which codes for several imprinted or biallelically expressed transcripts. The function of some of them are well understood (for example GSα-guanine nucleotide binding, α -stimulating protein is essential element of cell signaling), whereas the others are little known. The function of NESP55 (Neuroendocrine secretory protein 55) remains elusive, although there are suggestions about its role in brain development. Imprinted genes are currently being studied as potential candidate genes for quantitative trait loci (QTLs) in farm animals. In our study, we analyzed tissue distribution of NESP55 mRNA in pigs and established imprinting status of this gene in the brain stem, muscle, kidney and liver at several developmental stages. NESP55 mRNA was most abundant in central nervous system (CNS) and pituitary. Substantial expression was also noticed in the kidney, testis and muscle. Moreover, we identified a 12-nucleotides deletion within the coding region of NESP55 (accession number ss#342570450) which was used in imprinting analysis. The deletion was very rare in the analyzed populations and present only in heterozygous form. The imprinting analysis showed that NESP55 is maternally expressed in young and adult pigs, similar to what was obtained in humans, mice and cattle.