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.

Genetic Diversity and Population Structure of the Native Pulawska and Three Commercial Pig Breeds Based on Microsatellite Markers.

Swine DNA profiling is highly important for animal identification and parentage verification and also increasingly important for meat traceability. This work aimed to analyze the genetic structure and genetic diversity in selected Polish pig breeds. The study used a set of 14 microsatellite (STR) markers recommended by ISAG for parentage verification in the native Pulawska pig (PUL, n = 85) and three commercial pig breeds: Polish Large White (PLW, n = 74), Polish Landrace (PL, n = 85) and foreign breed Duroc (DUR, n = 84). Genetic differentiation among breeds accounted for 18% of the total genetic variability (AMOVA). Bayesian structure analysis (STRUCTURE) indicated that the four distinct genetic clusters obtained corresponded to the four breeds studied. The genetic Reynolds distances (Ɵw) showed a close relationship between PL and PLW breeds and the most distant for DUR and PUL pigs. The genetic differentiation values (FST) were lower between PL and PLW and higher between PUL and DUR. The principal coordinate analysis (PCoA) supported the classification of the populations into four clusters.

Genetic Structure of Racing Pigeons (Columba livia) Kept in Poland Based on Microsatellite Markers.

Pigeons played a major role in communication before the invention of the telephone and the telegraph, as well as in wars, where they were used to carry information and orders over long distances. Currently, numerous sports competitions and races are held with their participation, and their breeding is demanding not only for breeders, but also for the birds themselves. Therefore, an analysis of the genetic structure of racing pigeons kept in Poland was undertaken on the basis of 16 microsatellite markers, as well as the evaluation of the microsatellite panel recommended by ISAG. For this purpose, Bayesian clustering, a dendrogram, and Principal Coordinate Analysis were conducted. In addition, statistical analysis was performed. Based on this research, it was observed that racing pigeons are genetically mixed, regardless of their place of origin. Moreover, genetic diversity was estimated at a relatively satisfactory level (Ho = 0.623, He = 0.684), and no alarmingly high inbreeding coefficient was observed (F = 0.088). Moreover, it was found that the panel recommended by ISAG can be successfully used in Poland for individual identification and parentage testing (PIC = 0.639, CE-1P = 0.9987233, CE-2P = 0.9999872, CE-PP = 0.99999999).

How do genetic relatedness and spatial proximity shape African swine fever infections in wild boar?

The importance of social and spatial structuring of wildlife populations for disease spread, though widely recognized, is still poorly understood in many host-pathogen systems. In particular, system-specific kin relationships among hosts can create contact heterogeneities and differential disease transmission rates. Here, we investigate how distance-dependent infection risk is influenced by genetic relatedness in a novel host-pathogen system: wild boar (Sus scrofa) and African swine fever (ASF). We hypothesized that infection risk would correlate positively with proximity and relatedness to ASF-infected individuals but expected those relationships to weaken with the distance between individuals due to decay in contact rates and genetic similarity. We genotyped 323 wild boar samples (243 ASF-negative and 80 ASF-positive) collected in north-eastern Poland in 2014-2016 and modelled the effects of geographic distance, genetic relatedness and ASF virus transmission mode (direct or carcass-based) on the probability of ASF infection. Infection risk was positively associated with spatial proximity and genetic relatedness to infected individuals with generally stronger effect of distance. In the high-contact zone (0-2 km), infection risk was shaped by the presence of infected individuals rather than by relatedness to them. In the medium-contact zone (2-5 km), infection risk decreased but was still associated with relatedness and paired infections were more frequent among relatives. At farther distances, infection risk further declined with relatedness and proximity to positive individuals, and was 60% lower among un-related individuals in the no-contact zone (33% in10-20 km) compared among relatives in the high-contact zone (93% in 0-2 km). Transmission mode influenced the relationship between proximity or relatedness and infection risk. Our results indicate that the presence of nearby infected individuals is most important for shaping ASF infection rates through carcass-based transmission, while relatedness plays an important role in shaping transmission rates between live animals.

An inter-laboratory study of DNA-based identity, parentage and species testing in animal forensic genetics.

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel's proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Differentiating Pigs from Wild Boars Based on NR6A1 and MC1R Gene Polymorphisms.

This preliminary study aimed to differentiate domestic pigs from wild boars based on MC1R and NR6A1 polymorphisms and to identify admixture between these genomes. We studied samples obtained from wild boars from two regions of Poland and five pig breeds: Polish Landrace, Polish Large White, Zlotnicka White, Pulawska and Duroc. Along the MC1R gene sequence, we identified four polymorphic loci comprising three codons. The "wild type" allele was primarily found in wild boar but also in the Duroc and Zlotnicka White breeds. Non-wild type alleles were identified in the vast majority of domestic pig samples and in two wild boar samples. Based on MC1R profiles, we conducted a population study, and revealed admixture between both genomes using STRUCTURE and NETWORK Software. Interestingly, an allelic discrimination assay with NR6A1 g.748C > T TaqMan probes revealed a clear separation of samples into two groups: wild boar samples representing the C allele and domestic breeds representing the T allele. Based on the obtained results, we conclude that NR6A1 g.748C > T is an effective marker for differentiating between wild boars and domestic pigs, where this is supported by MC1R data, to identify admixed profiles. We recommend that a larger sample of genomes is studied to verify this method.

Microsatellite DNA Analysis for Diversity Study, Individual Identification and Parentage Control in Pig Breeds in Poland.

Swine DNA profiling is of high importance for animal identification and parentage verification. The aim of this study was to test a set of 14 microsatellite (STR) markers recommended by ISAG for parentage verification in Polish Landrace (PL, n = 900), Polish Large White (PLW, n = 482), Pulawska (PUL, n = 127), and Duroc pigs (DU n = 108). The studied breeds showed a medium level of genetic differentiation. The average value of heterozygosity and degree of polymorphism (PIC) were above 0.5 for the studied breeds, except for the DU breed (PIC = 0.477). The population inbreeding coefficient indicates an absence of inbreeding in the studied breeds (an average value of FIS = 0.007). The cumulative power of discrimination for all breeds reached high values close to 1.0, while the probability of identity (PID) was low, with PID values ranging between 10-9 (for DU) and 10-12 (for PLW). The cumulative exclusion probability for PE1 and PE2 showed that the parentage can be confirmed with a probability of from 92.75% to 99.01% and from 99.49% to 99.97%, respectively.

Microsatellite DNA Analysis of Genetic Diversity and Parentage Testing in the Popular Dog Breeds in Poland.

There is growing concern that extreme breed standardization contributes to a reduction of the effective population size and high levels of inbreeding, resulting in the loss of genetic diversity in many breeds. This study examined genetic diversity among eight popular dog breeds in Poland and evaluated the effectiveness of a 21-microsatellite (STR) panel recommended by the International Society for Animal Genetics (ISAG) for parent verification. The following breeds were characterized: German Shepherd, Maltese, Irish Wolfhound, Yorkshire Terrier, Biewer Yorkshire Terrier, Golden Retriever, Labrador Retriever, and French Bulldog. STRUCTURE analysis showed breed distinctiveness among all the dog breeds under study. Reynold's distance ranged between θw = 0.634 and θw = 0.260. The studied breeds showed a medium level of genetic differentiation; the mean number of alleles per locus ranged from 3.4 to 6.6, and the effective number of alleles from 2.1 to 3.5. The mean degree of heterozygosity varied from 49% to 69% and from 47% to 68% for HO and HE, respectively. The population inbreeding coefficient (FIS) indicated an absence of inbreeding in the studied breeds. The average polymorphism information content (PIC) values for most of the breeds were higher than 0.5. The cumulative power of discrimination (PD) for all the markers in all breeds reached high values (close to 1.0), while the probability of identity (PID) was low, ranging between 10-11 and 10-19. The cumulative exclusion probability when the genotypes of one (PE1) and both parents (PE2) are known and showed that the parentage can be confirmed with a probability of 94.92% to 99.95% and 99.78% to 99.9999%, respectively.

Pedigree and Molecular Analyses in the Assessment of Genetic Variability of the Polish Greyhound.

The aim of the study was to assess the genetic variability of the Polish Greyhound population based on pedigree analysis and molecular DNA testing and to determine the degree of relatedness among individuals in the population. Pedigree data of 912 Polish Greyhounds recorded in pedigree books since they were opened for this breed were analyzed. For molecular testing, DNA was obtained from cheek swabs taken from 235 dogs of the tested breed. A panel of 21 markers (Short Tandem Repeat-STR) was used. The mean inbreeding determined for the Polish Greyhound population based on pedigree analyses was low and amounted to 11.8%, but as many as 872 individuals of the 912 dogs in the studied population were inbred. A total of 83 founders (at least one unknown parent) were identified, among which 27 founders had both unknown parents. Full-sibling groups consisted of 130 individuals, with a minimum and maximum litter size of 2 and 16, respectively. The average litter size was 5.969. Gene diversity calculated based on the mean kinship matrix was 0.862 and the population mean kinship was 0.138. The founder genome equivalent based on the mean kinship matrix was 3.61; the founder genome surviving level was 12.34; the mean Ne was estimated at 21.76; and the Ne/N ratio was 0.135. The FIS inbreeding coefficient for 21 STR was negative, and the mean FIS value for all loci had a low negative value (-0.018). These values suggest a low level of inbreeding in the examined breed as well as the avoidance of mating related animals.

Fertile male tortoiseshell cat with true chimerism 38,XY/38,XY.

The tortoiseshell coat colour is characteristic to female cats, and its occurrence in tomcats is very rare and associated with chromosome abnormalities (additional copy of X chromosome). The aim of this study was identification of the genetic basis of a case of tortoiseshell colour in a fertile Maine coon tomcat. Cytogenetic and molecular genetic studies were carried out with painting molecular probes (WCPP) specific to the X and Y sex chromosomes as well as a DNA microsatellite panel for the parentage verification of cats. Cytogenetic analysis revealed only a single set of sex chromosomes typical for male - 38,XY. The results of the microsatellite polymorphism obtained from DNA showed three alleles in locus FCA201 and four alleles in loci FCA149 and FCA441 in different tissues (blood, hair roots and testicles). Based on these results, the case was diagnosed as a true chimerism 38,XY/38,XY. To the best of our knowledge, this is the first case of a 38,XY/38,XY chimera diagnosed in cats, confirmed by genetic analysis.

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.

The use of cytochrome b and ryanodine polymorphism to identify DNA of animal and human origin.

The aim of this study was to determine a match between DNA recovered from evidence material, such as knocked down red deer, and from comparative material in form of two brown traces on the bonnet of a car driven by a person suspected of knocking down the animal. The spots coming from the car provided no DNA profile, which questioned that they originated from a red deer and ruled out performance of a comparative DNA analysis. For this reason, the material obtained from the blood smear was analyzed for species identification. The method applied can discriminate between cattle, red deer and roe deer based on restriction analysis (Tsp509I) of PCR product (195bp), obtained by amplifying a fragment of the cytochrome b coding gene. Because the obtained restriction profile confirmed the match with red deer DNA for one trace, and in the second case ruled out that the biological traces originated from the species mentioned above, the PCR products were subjected to sequencing. In both cases, 195bp PCR products that were 98% homologous with red deer DNA sequence-NC_007704.2-trace1 and with the gene coding for the human ryanodine receptor-NC_008799.2-trace2. The quantity and quality of DNA obtained from the traces collected from the car bonnet did not allow confirmation of the involvement of a specific animal in the event, but the applied method made it possible to determine the species from which the obtained traces originated. Furthermore, the applied method, which was used earlier to determine cervine DNA, was successfully used to detect human DNA.

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.

Magnetization switching in ferromagnets by adsorbed chiral molecules without current or external magnetic field.

Ferromagnets are commonly magnetized by either external magnetic fields or spin polarized currents. The manipulation of magnetization by spin-current occurs through the spin-transfer-torque effect, which is applied, for example, in modern magnetoresistive random access memory. However, the current density required for the spin-transfer torque is of the order of 1 × 106 A·cm-2, or about 1 × 1025 electrons s-1 cm-2. This relatively high current density significantly affects the devices' structure and performance. Here we demonstrate magnetization switching of ferromagnetic thin layers that is induced solely by adsorption of chiral molecules. In this case, about 1013 electrons per cm2 are sufficient to induce magnetization reversal. The direction of the magnetization depends on the handedness of the adsorbed chiral molecules. Local magnetization switching is achieved by adsorbing a chiral self-assembled molecular monolayer on a gold-coated ferromagnetic layer with perpendicular magnetic anisotropy. These results present a simple low-power magnetization mechanism when operating at ambient conditions.

Genetic differentiation among 6 populations of red deer (Cervus elaphus L.) in Poland based on microsatellite DNA polymorphism.

Recently, there has been considerable interest in genetic differentiation in the Cervidae family. A common tool used to determine genetic variation in different species, breeds and populations is DNA analysis, which allows for direct determination of the differences and changes within a group of animals. Because the analysis of microsatellite polymorphism in different Cervidae populations revealed considerable genetic variability in individual populations, it was important to test a set of markers in animals from these populations.The study was performed with muscle tissue and blood samples collected from a total of 793 red deer. Six groups (subpopulations) of red deer were defined according to region: Masurian (330 animals), Bieszczady (194 animals), Malopolska (80 animals), Sudety (76 animals), Lower Silesian (62 animals) and Lubusz (51 animals). The analysis involved 12 STR markers (BM1818, OarAE129, OarFCB5, OarFCB304, RM188, RT 1, RT 13, T26, T156, T193, T501, TGLA53), for which conditions for simultaneous amplification were established.Based on this study, it is concluded that the chosen set of 12 microsatellite markers could be used to evaluate the genetic structure and to monitor changes in Poland's red deer population.

Electrochemically "writing" graphene from graphene oxide.

A novel approach of patterning graphene on conductive surfaces based on local electrochemical reduction of graphene oxide is reported. Graphene is "written" from typical graphene oxide dispersion by applying negative potential on conductive surfaces vs. a micrometer-sized counter electrode "pen" with scanning electrochemical microscopy (SECM). Micrometer scaled patterns are successfully generated on gold and stainless steel surfaces.

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.

Electrochemical codeposition of sol-gel films on stainless steel: controlling the chemical and physical coating properties of biomedical implants.

The electrochemically assisted codeposition of sol-gel thin films on stainless steel is described. Specifically, electrodeposition of films based on aminopropyltriethoxysilane (APTS), and its codeposition with propyltrimethoxysilane (PrTMOS) and phenyltrimethoxysilane (PhTMOS) has been accomplished by applying negative potentials. The latter increases the concentration of hydroxyl ions on the stainless steel surface and thus catalyzes the condensation and deposition of the sol-gel films. The films were characterized by profilometry, electrochemical impedance spectroscopy (EIS), alternating current voltammetry (ACV), goniometry, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM analysis of codeposited APTS:PrTMOS films disclosed the structural changes induced by altering the deposition solution composition and the applied potential. Codeposited APTS:PhTMOS did not show any structural differences from their electrodeposited homopolymers, while Nano Scratch Test clearly revealed the changes in the elastic and adhesion properties, suggesting the formation of an APTS:PhTMOS composite. EIS of the films showed good resistance towards penetration of hydrophilic species, such as hexacyanoferrate. ACV measurements of the homo and codeposits showed the decrease of the interfacial capacity as a result of the electrochemical deposition. In essence, controllable sol-gel films with tunable chemical and physical properties based on controlling the combination of the precursors, pH and electrochemical properties can be electrodeposited on conducting surfaces. The application of this approach has been demonstrated by coating a stainless steel coronary stent.

Characteristics of X- and Y-chromosome specific regions of the amelogenin gene and a PCR-based method for sex identification in red deer (Cervus elaphus).

The present study attempts to analyse sequences of the X- and Y-chromosome specific regions of the amelogenin (AMEL) gene in red deer. To this end, primers specific for each form of the gene (AMELX and AMELY) were designed based on bovine genomic sequences and the homologous regions of the genes were sequenced. The obtained sequence of AMELX gene showed high similarity with the corresponding region in cattle (91%) and humans (77%), but this similarity was slightly lower among AMELY genes and showed 87 and 73% of identical nucleotides, respectively. In addition, three single nucleotide polymorphisms (SNPs) were found in the AMELX gene of the female red deer investigated. Comparative analysis of the homologous fragments of the red deer AMELX and AMELY genes confirmed the deletion of an AMELY gene fragment in relation to AMELX. Homology of both sequences was 82% of identical nucleotides in the coding region and 74% in 3' non-coding sequence. The sequences studied showed considerable similarity to homologous fragments of the human and bovine gene, but the structural differences observed lead us to design PCR-based method for sex identification in red deer, based on the presented sequences.

Acid resistance of the enamel in primary second molars from children with down syndrome and cerebral palsy.

OBJECTIVES: This study was carried out to evaluate the extent of differences in mineralization of inner and outer enamel of the lower primary second molars of children with Down syndrome (DS) and Cerebral Palsy (CP) as revealed by acid treatment of exfoliated teeth. The results were compared to those obtained from a control group of healthy children. METHODS: The sample included 4 mandibular second molars from each group. On each tooth, a thin section was cut, bisecting the mesial cusps. The analysis was carried out on the mesio-buccal cusps. Atomic force microscopy (AMF) was used to analyze the morphological structure of the dental enamel after 10 sec of 0.1 mol% citric acid treatment. The measurements were performed on 3 points in the enamel close to the outer surface and 3 points in the enamel close to the dentin. The differences between groups were analyzed using Mann Whitney tests. RESULTS: In controls and CP teeth the outer enamel was more resistant to etching than the inner enamel. In DS teeth both outer and inner enamel showed similar results for all parameters. Between group comparisons showed that roughness values were significantly higher (P<0.01) in DS teeth than in either controls or CP teeth. No significant differences were found between CP and control teeth. CONCLUSIONS: The higher values obtained for DS enamel reflect increased solubility of the enamel to acid relative to controls and CP teeth together with irregularity of the organic matrix. The practical importance of the results is that DS primary molars needs reduced etching time when prepared for pit and fissure sealants or composite/compomer restorations.