In Silico Analysis of Seven PCR Markers Developed from the CHD1, NIPBL and SPIN Genes Followed by Laboratory Testing Shows How to Reliably Determine the Sex of Musophagiformes Species.

Sex determination in birds, due to the very common lack of sexual dimorphism, is challenging. Therefore, molecular sexing is often the only reliable way to differentiate between the sexes. However, for many bird species, very few genetic markers are available to accurately, quickly, and cost-effectively type sex. Therefore, in our study, using 14 species belonging to the order Musophagiformes, we tested the usefulness of seven PCR markers (three of which have never been used to determine the sex of turacos), developed based on the CHD1, NIPBL, and SPIN genes, to validate existing and develop new strategies/methods of sex determination. After in silico analysis, for which we used the three turaco nuclear genomes available in GenBank, the suitability of the seven selected markers for sexing turacos was tested in the laboratory. It turned out that the best of the markers tested was the 17th intron in the NIPBL gene (not previously tested in turacos), allowing reliable sex determination in 13 of the 14 species tested. For the one species not sexed by this marker, the 9th intron in the CHD1 gene proved to be effective. The remaining markers were of little (4 markers developed based on the CHD1 gene) or no use (marker developed based on the SPIN gene).

The Length Polymorphism of the 9th Intron in the Avian CHD1 Gene Allows Sex Determination in Some Species of Palaeognathae.

In palaeognathous birds, several PCR-based methods and a range of genes and unknown genomic regions have been studied for the determination of sex. Many of these methods have proven to be unreliable, complex, expensive, and time-consuming. Even the most widely used PCR markers for sex typing in birds, the selected introns of the highly conserved CHD1 gene (primers P2/P8, 1237L/1272H, and 2550F/2718R), have rarely been effective in palaeognathous birds. In this study we used eight species of Palaeognathae to test three PCR markers: CHD1i9 (CHD1 gene intron 9) and NIPBLi16 (NIPBL gene intron 16) that performed properly as Psittaciformes sex differentiation markers, but have not yet been tested in Palaeognathae, as well as the CHD1iA intron (CHD1 gene intron 16), which so far has not been used effectively to sex palaeognathous birds. The results of our research indicate that the CHD1i9 marker effectively differentiates sex in four of the eight species we studied. In Rhea americana, Eudromia elegans, and Tinamus solitarius, the electrophoretic patterns of the amplicons obtained clearly indicate the sex of tested individuals, whereas in Crypturellus tataupa, sexing is possible based on poorly visible female specific bands. Additionally, we present and discuss the results of our in silico investigation on the applicability of CHD1i9 to sex other Palaeognathae that were not tested in this study.

Usefulness of Selected Acute-Phase Proteins in the Postsurgical Monitoring of Arthroscopy and Splint Bone Removal in Horses.

BACKGROUND: Arthroscopy and splint bone removal are the common orthopedic procedures in horses. Estimation of the dynamics of acute phase proteins in postoperative monitoring seems to be interesting diagnostic approach. The aim of the study was to investigate changes in the concentrations of plasma inflammatory markers-fibrinogen, haptoglobin, and protease inhibitors-following orthopedic surgery in horses. The study involved 114 horses, divided into two study groups undergoing: arthroscopy (41 horses) and splint bone removal (13 horses). The control group consisted of 60 healthy horses. The blood was collected before the surgery and 24, 48, 72 h, 5, 7, 10, 14 and 28 days after the surgery. Plasma fibrinogen, serum haptoglobin and proteinase inhibitors were measured. RESULTS: In non-complicated cases of arthroscopy and splint bone removal, fibrinogen and haptoglobin increased stepwise from 24 h, achieved the maximum level at 72 h and returned to preoperative levels after 10-14 days. In one complicated case after arthroscopy surgery the marked increase in fibrinogen and haptoglobin concentrations was observed 24 h earlier than standard parameters of inflammation Conclusion: The study shows the evolution of APPs after arthroscopy and splint bone removal in 28 days postsurgery period and in the case of one complicated case of arthroscopy.

New Bird Sexing Strategy Developed in the Order Psittaciformes Involves Multiple Markers to Avoid Sex Misidentification: Debunked Myth of the Universal DNA Marker.

Sexing of birds is indispensable for scientific, breeding and conservation programs but is difficult in many species and is particularly problematic in the case of nestlings showing no sexual dimorphism. Most useful and efficient methods of sex determination are based on unique features of the Z and W sex chromosomes detected via PCR to distinguish males (ZZ) and females (ZW). During the last twenty-five years researchers searched for the universal marker capable of sexing a maximally wide spectrum of species in a single PCR assay. We screened the phylogenetically representative set of 135 Psittaciformes species including 59 species sexed for the first time. Two known (P2P8, CHD1iA) PCR markers and four additional W/Z polymorphisms (CHD1iE, CHD1i16, CHD1i9 and NIPBLi16) located within the Chromo Helicase DNA binding CHD1 or the Nipped-B homolog NIPBL genes were applied. We present the electrophoretic patterns obtained for the PCR products of the analyzed markers including most typical and atypical patterns allowing sex determination, as well as those obtained when the given marker failed in sexing. Technical aspects of molecular sex determination are discussed: the optimization of amplification conditions, direct PCR and potential misinterpretations. A truly universal marker has not been found, and therefore, we propose a sexing strategy based on multiple CHD1i16, NIPBLi16, CHD1i9 and CHD1iE markers. This new strategy confirms the sex of a given bird with at least two markers detecting independent Z/W polymorphisms, reduces the number of necessary PCR reactions and minimizes the risk of sex misidentification.

Late Pleistocene Expansion of Small Murid Rodents across the Palearctic in Relation to the Past Environmental Changes.

We investigated the evolutionary history of the striped field mouse to identify factors that initiated its past demographic changes and to shed light on the causes of its current genetic structure and trans-Eurasian distribution. We sequenced mitochondrial cyt b from 184 individuals, obtained from 35 sites in central Europe and eastern Mongolia. We compared genetic analyses with previously published historical distribution models and data on environmental and climatic changes. The past demographic changes displayed similar population trends in the case of recently expanded clades C1 and C3, with the glacial (MIS 3-4) expansion and postglacial bottleneck preceding the recent expansion initiated in the late Holocene and were related to environmental changes during the upper Pleistocene and Holocene. The past demographic trends of the eastern Asian clade C3 were correlated with changes in sea level and the formation of new land bridges formed by the exposed sea shelf during the glaciations. These data were supported by reconstructed historical distribution models. The results of our genetic analyses, supported by the reconstruction of the historical spatial distributions of the distinct clades, confirm that over time the local populations mixed as a consequence of environmental and climatic changes resulting from cyclical glaciation and the interglacial period during the Pleistocene.

Insight into the Genetic Population Structure of Wild Red Foxes in Poland Reveals Low Risk of Genetic Introgression from Escaped Farm Red Foxes.

In this study we assessed the level of genetic introgression between red foxes bred on fur farms in Poland and the native wild population. We also evaluated the impact of a geographic barrier and isolation by distance on gene flow between two isolated subpopulations of the native red fox and their genetic differentiation. Nuclear and mitochondrial DNA was collected from a total of 308 individuals (200 farm and 108 wild red foxes) to study non-native allele flow from farm into wild red fox populations. Genetic structure analyses performed using 24 autosomal microsatellites showed two genetic clusters as being the most probable number of distinct populations. No strong admixture signals between farm and wild red foxes were detected, and significant genetic differentiation was identified between the two groups. This was also apparent from the mtDNA analysis. None of the concatenated haplotypes detected in farm foxes was found in wild animals. The consequence of this was that the haplotype network displayed two genetically distinct groups: farm foxes were completely separated from native ones. Neither the River Vistula nor isolation by distance had a significant impact on gene flow between the separated wild red fox subpopulations. The results of our research indicate a low probability of genetic introgression between farm and native red foxes, and no threat to the genetic integrity of this species.

How Selective Breeding Has Changed the Morphology of the American Mink (Neovison vison)-A Comparative Analysis of Farm and Feral Animals.

In this study, we performed a comparative analysis of the morphological traits between feral (n = 43) and farm (n = 200) individuals of the American mink in Poland to address the question of how multigenerational intensive selective breeding has morphologically differentiated these two populations. Nine body measurements and two proportion coefficients were obtained using adult individuals. The significance of differences between population means was assessed using the Wilcoxon test for independent samples, while the Kruskal-Wallis test was used to compare sex-population groups. Spearman's correlation coefficients between measurements were estimated for each population. We also performed Principal Component Analysis (PCA) to identify the variables that were most closely correlated with variation in the trait measurements and to investigate the morphological differences between farm and feral minks. We found that the farm minks exhibited significantly higher mean values for eight out of eleven studied traits. Moreover, significant changes in forelimb length, with no concomitant changes in hindlimb length, were accompanied by differences in body shape: trapezoidal in feral minks and rectangular in farm minks. The PCA suggested an almost complete separation of the two populations and indicated that sexes were quite separate; farm males in particular constitute a wholly discrete cluster. Such a clear differentiation between the two populations and sexes over a period of several decades highlights the intensity of selective breeding in shaping the morphology of these animals.

Admixture analyses and phylogeographic relationships reveal complete genetic distinctiveness of Polish farm and wild red foxes (Vulpes vulpes) and the North American origin of farm-bred individuals.

A number of studies showed that many mtDNA haplotypes were shared among contemporary farm red foxes bred on different continents and the historical wild red foxes of North American origin. Therefore, in this study, the population genetic structure and phylogeographic relationships of Polish red foxes kept on fur farms and their wild conspecifics were investigated to assess the ancestry of the farm red foxes in Poland. A total of 330 tissue samples (200 from farm foxes and 130 from wild foxes) were used for the genetic analyses. Thirty microsatellite loci and two regions of mtDNA were used to assess the level of admixture between farm- and wild red foxes, to construct haplotype networks and create a phylogenetic tree. The genetic structure analysis clearly indicated two genetic clusters as being the most probable number of genetically distinct populations. The fixation index revealed a significant genetic distance between the farm- and wild red fox populations (FST  = 0.27, p < 0.05). Haplotype networks based on frequencies showing relationships between concatenated haplotypes of Polish farm- and wild red foxes and the constructed phylogenetic tree clearly indicated two genetically distinct groups. The results of this study provide strong evidence confirming the North American origin of red foxes bred on Polish farms and the genetic distinctiveness of both studied populations.

High-frequency marker haplotypes in the genomic selection of dairy cattle.

The aim of this study was to predict the genomic breeding value (DGV) of production, selected conformation and reproductive traits, and somatic cell score of dairy cattle in Poland using high-frequency marker haplotypes. The dataset consisted of phenotypic, genotypic, and pedigree data of 1216 Polish Holstein-Friesian bulls. The genotypic data consisted of 54,000 single-nucleotide polymorphisms (SNPs). The data were divided into two subsets: a test dataset (n = 1064) and a validation dataset (n = 152). Genotypic data were selected using three criteria: the percentage of missing genotypes, minor allele frequency, and linkage disequilibrium. The purpose of the data selection was to identify blocks of SNPs that were then used for the construction of haplotypes. Only haplotypes with a frequency higher than 25% were selected. DGV was predicted using four variants of a linear model with random haplotype effects and deregressed breeding values as the response variables. The accuracy of genomic prediction was checked by comparing DGVs with estimated breeding values (EBVs) using two methods: Pearson's correlations and the regression of EBV on DGV. The use of high-frequency haplotypes showed a tendency to underestimate DGVs. None of the models tested was clearly superior with regard to the traits studied. DGVs of production and conformation traits as well as somatic cell score (medium or high heritability traits) were more accurate than those estimated for fertility traits (low heritability traits).

Evolutionary history and phylogeographic relationships of shrews from Sorex araneus group.

Shrews of the Sorex genus are an evolutionarily successful group that includes more than 77 species widely distributed in Eurasia and North America. The genus is one of the rare cases where karyotypic changes reflect well the evolutionary relationships among its species. The taxa showing the greatest variation in karyotype are usually classified into the Sorex araneus group. Its evolution was associated with chromosomal rearrangements, which could have promoted fast diversification of this group into many chromosomal races and species. These processes were additionally complicated by introgressions of mitochondrial DNA, which made the evolutionary history of this group quite complex and difficult to infer. To tackle the problem, we performed multi-method phylogenetic analyses based on mitochondrial cytochrome b that is considered a good molecular marker available for many representatives of Sorex. The results were compared with phylogenies based on chromosomal rearrangement data and put into temporal and spatial context using molecular dating and historical biogeography methods. We complemented the study with the estimation of diversification rates within the S. araneus group as well as comparing the results with paleontological records and climatic oscillations within the last 4 million years. Based on the gathered data, we proposed a hypothetical scenario for the evolution and geographic dispersion of species belonging to the S. araneus group. The shrews began to diversify about 2.7 million years ago in Eurasia and then migrated at least twice to North America. The evolution of shrews was driven by Pleistocene glacial and interglacial cycles, which increased their speciation rate and the emergence of new lineages. The migrations of populations were accompanied by introgressions of mitochondrial DNA into native shrews and occurred at least twice.

Microsatellite polymorphism and its association with body weight and selected morphometrics of farm red fox (Vulpes vulpes L.).

Polymorphism of 30 canine-derived microsatellites was studied in a group of 200 red foxes kept on 2 Polish farms. 22 out of 30 microsatellites were selected to study association between marker genotypes and body weight (BW), body length (BL), body circumference (BC), tail length (TL), ear height (EH), length of the right front limb (FRLL), length of the right rear limb (RRLL), length of the right front foot (FRFL) and length of the right rear foot (RRFL). A total of 112 alleles and 243 genotypes were found at 22 autosomal microsatellite loci. Three monomorphic loci deemed as uninformative were excluded from the study. The association between marker genotypes and the studied traits was analysed using general linear model (GLM) procedure and least squares means (LSM). Linkage disequilibrium (LD) was estimated to assess non-random association between microsatellite loci. Out of 19 microsatellites studied four markers showed no association with the studied traits, three markers had a significant effect on one trait, and another three markers had significant effect on two traits. Among ten microsatellites with significant effect on four economically important traits (BW, BL, BC, TL) four were associated with two characters: marker FH2613 with BW and BC, marker FH2097withBL and BC, marker ZUBECA6 with BW and BC, whereas marker REN75M10 was associated with BL and TL. The strongest LD (r(2) ranged from 0.15 to 0.33) was estimated between nine loci with significant effect on economically important traits (BW, BL, BC, TL).

Linear models for breeding values prediction in haplotype-assisted selection - an analysis of QTL-MAS Workshop 2011 Data.

BACKGROUND: The aim of this study was to estimate haplotype effects and then to predict breeding values using linear models. The haplotype based analysis enables avoidance of loosing information due to linkage disequilibrium between single markers. There are also less explanatory variables in the linear model which makes the estimation more reliable. METHODS: Different methods and criteria for marker and haplotype selection were considered. First, markers with MAF lower than 5% where excluded from the data set. Then, SNPs in complete linkage disequilibrium where selected. Next step was to construct haplotypes and to estimate their frequencies basing on selected SNPs. The haplotypes with a frequency lower than 1% were not considered in further analysis. Chosen haplotypes were used as the explanatory variables in the linear models for breeding values prediction. Linear models with fixed and random haplotype effects as well as animal model were tested. RESULTS: The number of markers was limited to 1206, 1189, 1249, 1288 and 1167 for chromosome 1, 2, 3, 4 and 5, respectively due to MAF criterion. In total 409 subsets of SNPs with r2=1 were found. 1476 haplotypes with different lengths were inferred. The frequencies of 817 haplotypes were higher than 1% - 184 for the first chromosome, 172 for the second, 131 for the third, 146 for the forth and 184 haplotypes for the fifth chromosome. The haplotype effects estimated using random models were comparable and more precise in prediction for individuals with unknown phenotypes. A few haplotypes with large effects were found when their effects were defined as fixed in the linear model . The correlations of the predicted breeding values with true breeding values were not that high. This could be brought about by selection criteria imposed on the genotype data which led to substantial reduction of number of markers. CONCLUSIONS: Although not many markers were considered in the study, the results obtained show that the implemented approach can be considered as quite promising. The haplotype approach let to avoid high dimensional models as compared with single SNPs models.

A microsatellite study in the Legucki Mlyn/Popielno hybrid zone reveals no genetic differentiation between two chromosome races of the common shrew (Sorex araneus).

This study investigated a chromosome hybrid zone between two chromosomal races of the common shrew (Sorex araneus). Gene flow and genetic structure of the hybrid zone, located in the northeast of Poland, were studied using seven polymorphic autosomal microsatellite loci (L9, L14, L33, L45, L67, L68, L97) and a Y-linked microsatellite locus (L8Y). Seventy-five animals (46 of the Legucki Mlyn race and 29 of the Popielno race) from nine different localities were examined and the data were analyzed using hierarchical AMOVA and F-statistic. The studied microsatellite loci and races (divided into nine geographical populations) were characterized by observed heterozygosity (H(O)), expected heterozygosities within (H(S)), and between (H(T)) populations, inbreeding coefficient (F(IS)), fixation index (F(ST)), and average allelic richness (A). We found that genetic structuring within and between the two chromosome races were weak and non-significant. This finding and unconstrained gene flow between the races indicates a high level of migration within the Legucki Mlyn/Popielno hybrid zone, suggesting that evolutionarily important genetic structuring does not occur in interracial zones where races which are not genetically distinct come into contact.

Statistical aspects of genetic association testing in small samples, based on selective DNA pooling data in the arctic fox.

We analysed data from a selective DNA pooling experiment with 130 individuals of the arctic fox (Alopex lagopus), which originated from 2 different types regarding body size. The association between alleles of 6 selected unlinked molecular markers and body size was tested by using univariate and multinomial logistic regression models, applying odds ratio and test statistics from the power divergence family. Due to the small sample size and the resulting sparseness of the data table, in hypothesis testing we could not rely on the asymptotic distributions of the tests. Instead, we tried to account for data sparseness by (i) modifying confidence intervals of odds ratio; (ii) using a normal approximation of the asymptotic distribution of the power divergence tests with different approaches for calculating moments of the statistics; and (iii) assessing P values empirically, based on bootstrap samples. As a result, a significant association was observed for 3 markers. Furthermore, we used simulations to assess the validity of the normal approximation of the asymptotic distribution of the test statistics under the conditions of small and sparse samples.