Abstract Background With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. Results Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. Conclusions The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

Several genetic variants have been shown to affect the mean number of offspring in different sheep breeds. Here, we analyzed samples from Icelandic sheep with the aim of identifying the genetic cause of the Icelandic Loa phenotype using three previously identified prolificacy genes as candidates. We demonstrate that a 4-bp frameshift deletion positioned in the mature region of the GDF9 protein in the Loa animals is a likely causal mutation for the observed increase in prolificacy; however, sequencing showed that not all ewes with a high number of offspring carried the deletion, suggesting the presence of a second mutation segregating within this group of animals.

BackgroundThe plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce.ResultsOur phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)and three-LU domain containing genes in generaloccurred later in evolution and was first detectable after coelacanths.ConclusionsThis study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several membershitherto unknown in mammalsprovide new perspectives on the evolution of this important enzyme system.

Stature is affected by many polymorphisms of small effect in humans 1 . In contrast, variation in dogs, even within breeds, has been suggested to be largely due to variants in a small number of genes2,3. Here we use data from cattle to compare the genetic architecture of stature to those in humans and dogs. We conducted a meta-analysis for stature using 58,265 cattle from 17 populations with 25.4 million imputed whole-genome sequence variants. Results showed that the genetic architecture of stature in cattle is similar to that in humans, as the lead variants in 163 significantly associated genomic regions (P < 5 × 10−8) explained at most 13.8% of the phenotypic variance. Most of these variants were noncoding, including variants that were also expression quantitative trait loci (eQTLs) and in ChIP–seq peaks. There was significant overlap in loci for stature with humans and dogs, suggesting that a set of common genes regulates body size in mammals. Meta-analysis of data from 58,265 cattle shows that the genetic architecture underlying stature is similar to that in humans, where many genomic regions individually explain only a small amount of phenotypic variance.

The American mink (Neovison vison) is a semiaquatic mustelid native to North America. It is an important animal for the fur industry. Although many efforts have been made to locate genes influencing fur quality and color, the lack of a reference genome impedes the search. American mink has the smallest chromosome number among studied Carnivora species. Genomic information about American mink is also vital to understand the evolution of Carnivora . Hence a reference genome of mink will facilitate genetic improvement of economic traits and will increase our knowledge about the evolution of Carnivora . Here we present the draft genome sequence of American mink. In our study, a male inbred pearl mink was sequenced by Illumina paired-end and mate pair sequencing. The reads were assembled, which lead to 22,419 scaffolds with an N50 (shortest sequence length at 50% of the genome) of 646,304 bp. The assembly constituted 2.4G plus gaps, representing 90% of the estimated genome size. Repeat annotation showed that repeat sequences constitute about 25% of the mink genome. The biggest repeat family was a family of LINEs similar to LINEs found in the dog and ferret genomes. Gene annotation of our draft genome indicated our draft genome contains 87% of 1:1 Vertebrata genes (63.5% complete single copy genes, 0.5% duplicated genes and 22% fragment genes). We were able to map on the draft genome all the well-studied genes which are thought to be involved in the coat quality and coat color phenotypes. Our draft genome has great potential to facilitate genomic research towards improved breeding for high fur quality and will strengthen our understanding of Carnivora evolution.

Summary Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin super-family and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central β–sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI–1 variants undergo latency transition with a similar rate. Since the functional stability of PAI–1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system. Supplementary Material to this article is available online at www.thrombosis-online.com.

During myogenesis the satellite cells undergo a series of highly ordered events that include division and proliferation of mononucleated myoblasts, irreversible cell cycle withdrawal, elongation of myoblasts and finally cell-cell fusion, leading to the formation of multinucleated myotubes. These cellular processes are strictly regulated in a spatiotemporally manner through a coordinated patterns of gene expression governed by specific transcriptional activators and networks of signal transduction. Here we analyzed the genome-wide changes in gene transcription occurring during growth and differentiation of porcine primary myoblast cells. Analysis for differential expression revealed 1656 gene transcripts with significant changes in expression levels during myogenesis. These analyses expand our knowledge of the temporal expression patterns and provide an insight into known and unknown genes and pathways that are transcriptionally regulated and, therefore, likely to be critically implicated in myogenesis. Intriguingly, a remarkable number of the differentially expressed genes encode proteins engaged in histone modification and remodeling of nucleosome structure, emphasizing that extensive changes in chromatin structure are required for regulated gene transcription in the transition from proliferating myoblasts to terminally differentiated myotubes.

The effect on rennet-induced milk coagulation of three novel genetic haplotypes in close proximity to CSN3 encoding κ-casein was evaluated. Milk samples were collected from 71 Danish Holstein cows homozygous for the three novel haplotypes named according to which genetic variants of CSN3 they were characterised by: AE, A and B, respectively. The results documented that haplotype AE had significantly longer rennet coagulation time and lower curd firming rate compared with haplotypes A and B. Haplotype AE milk was further characterised by larger casein micelles and lower relative content of κ-casein, whereas the total protein contents did not differ among haplotypes. These findings indicate that the genetic κ-casein A variant can be divided into two groups with poor and good milk coagulation properties. Furthermore, three milk samples were identified as non-coagulating. These were all associated with the haplotype AE.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

The American mink (Neovison vison) is a semiaquatic species of mustelid native to North America. It's an important animal for the fur industry. Many efforts have been made to locate genes influencing fur quality and color, but this search has been impeded by the lack of a reference genome. Here we present the first draft genome of mink. In our study, two mink individuals were sequenced by Illumina sequencing with 797 Gb sequence generated. Assembly yielded 7,175 scaffolds with an N50 of 6.3 Mb and length of 2.4 Gb including gaps. Repeat sequences constitute around 31% of the genome, which is lower than for dog and cat genomes. The alignments of mink, ferret and dog genomes help to illustrate the chromosomes rearrangement. Gene annotation identified 21,053 protein-coding sequences present in mink genome. The reference genome's structure is consistent with the microsatellite-based genetic map. Mapping of well-studied genes known to be involved in coat quality and coat color, and previously located fur quality QTL provide new knowledge about putative candidate genes for fur traits. The draft genome shows great potential to facilitate genomic research towards improved breeding for high fur quality animals and strengthen our understanding on evolution of Carnivora.

The HTR2C gene encodes the 5-Hydroxytryptamine (serotonin) receptor 2C, G-protein-coupled protein which functions as a serotonin receptor. The HTR2C mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2). In the current study we examined the molecular characteristics of the porcine HTR2C gene and determined the mRNA editing of the HTR2C transcript in different tissues. The A-to-I RNA editing of HTR2C was shown to be conserved in the porcine homologue with five nucleotides edited in exon 5. A differential editing was demonstrated with a high editing frequency in the frontal cortex, parietal cortex, occipital cortex, hypothalamus, brain stem and spinal cord and significantly lower in the cerebellum. No editing was seen in the liver and kidney. The porcine HTR2C gene was found to be exclusively expressed in brain tissues. The HTR2C gene was mapped to pig chromosome X. The methylation status of the HTR2C gene was examined in brain and liver by bisulfate sequencing and a high degree of methylation was found in the two tissues, at 89 and 72%, respectively. Our data describe differences in RNA editing in various regions of the porcine brain. The differences might reflect functional differences. Similarities between pigs and humans in differential RNA editing support the use of the pig as a model organism for the study of neurological diseases.

The IGFBP7 gene encodes insulin-like growth factor protein 7. IGFPB7 is involved in diverse biological functions including cell growth regulation, senescence and apoptosis, and also acts as a tumor suppressor in multiple cancers. The IGFBP7 mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2). In the current study we have examined molecular characteristics of the porcine IGFBP7 gene, and determined the mRNA editing in different tissues. The A-to-I RNA editing of human IGFBP7 in positions Arg78 and Lys95 was shown to be conserved in the porcine homologue. In addition, a novel editing site was discovered in position Lys97 in the porcine IGFBP7 transcript. A differential editing was demonstrated at the three positions in the IGFBP7 transcript with very high degrees of editing in frontal cortex, cerebellum and lung. Interestingly, the degree of editing increased during aging in porcine frontal cortex and cerebellum. The IGFBP7 gene was mapped to pig chromosome 8. The porcine IGFBP7 gene was found to be ubiquitously expressed in examined organs and tissues. The methylation status of the IGFBP gene was examined in brain and liver by bisulfate sequencing and a high degree of methylation was found in the two tissues, 52% and 54%, respectively.

Rennet-induced milk coagulation is an important trait for cheese production. Recent studies have reported an alarming frequency of cows producing poorly coagulating milk unsuitable for cheese production. Several genetic factors are known to affect milk coagulation, including variation in the major milk proteins; however, recent association studies indicate genetic effects from other genomic regions as well. The aim of this study was to detect genetic variation affecting milk coagulation properties, measured as curd-firming rate (CFR) and milk pH. This was achieved by examining allele frequency differences between pooled whole-genome sequences of phenotypically extreme samples (pool-seq).. Curd-firming rate and raw milk pH were measured for 415 Danish Holstein cows, and each animal was sequenced at low coverage. Pools were created containing whole genome sequence reads from samples with "extreme" values (high or low) for both phenotypic traits. A total of 6,992,186 and 5,295,501 SNP were assessed in relation to CFR and milk pH, respectively. Allele frequency differences were calculated between pools and 32 significantly different SNP were detected, 1 for milk pH and 31 for CFR, of which 19 are located on chromosome 6. A total of 9 significant SNP, which were selected based on the possible function of proximal candidate genes, were genotyped in the entire sample set ( = 415) to test for an association. The most significant SNP was located proximal to , explaining 33% of the phenotypic variance. , coding for κ-casein, is the most studied in relation to milk coagulation due to its position on the surface of the casein micelles and the direct involvement in milk coagulation. Three additional SNP located on chromosome 6 showed significant associations explaining 7, 3.6, and 1.3% of the phenotypic variance of CFR. The significant SNP on chromosome 6 were shown to be in linkage disequilibrium with the SNP peaking proximal to ; however, after accounting for the genotype of the peak SNP within this QTL, significant effects (-value < 0.1) could still be detected for 2 of the SNP accounting for 2 and 1% of the phenotypic variance. These 2 interesting SNP were located within introns or proximal to the candidate genes-solute carrier family 4 (sodium bicarbonate cotransporter), member 4 () and LIM and calponin homology domains 1 (), respectively-making them interesting targets for further analysis.

Orphan receptor NURR1 (also termed NR4A2) belongs to the nuclear receptor superfamily and functions as a regulatory factor of differentiation, migration, maturation and maintenance of mesencephalic dopaminergic neurons. NURR1 plays an important role in nigrostriatal dopamine neuron development and is therefore implicated in the pathogenesis of neurodegenerative diseases linked to the dopamine system of the midbrain. Here we report the isolation and characterization of porcine NURR1 cDNA. The NURR1 cDNA was RT-PCR cloned using NURR1-specific oligonucleotide primers derived from in silico sequences. The porcine NURR1 cDNA encodes a polypeptide of 598 amino acids, displaying a very high similarity with bovine, human and mouse (99%) NURR1 protein. Expression analysis revealed a differential NURR1 mRNA expression in various organs and tissues. NURR1 transcripts could be detected as early as at 60 days of embryo development in different brain tissues. A significant increase in NURR1 transcript in the cerebellum and a decrease in NURR1 transcript in the basal ganglia was observed during embryo development. The porcine NURR1 gene was mapped to chromosome 15. Two missense mutations were found in exon 3, the first coding exon of NURR1. Methylation analysis of the porcine NURR1 gene body revealed a high methylation degree in brain tissue, whereas methylation of the promoter was very low. A decrease in DNA methylation in a discrete region of the NURR1 promoter was observed in pig frontal cortex during pig embryo development. This observation correlated with an increase in NURR1 transcripts. Therefore, methylation might be a determinant of NURR1 expression at certain time points in embryo development.

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

Honey bee workers were sampled across the Serbian territory during 2009-2010 from mostly non-migratory apiaries to determine the population structure of these bees using morphometric, genetic, and spatial information. A total of 134 bees were sampled, of which 77 were analysed using classical wing morphometrics and 122 bees were successfully analysed using 24 DNA microsatellite markers. A combination of methods including multivariate statistics and assignment tests (frequency-based and Bayesian) revealed the honey bees of this region to resemble the subspecies Apt mellifera macedonica, Apis mellifera carnica or hybrids of these two subspecies. Based on Bayesian assignment ('Structure') and spatial PCA, honey bees within the Serbian territory were composed of 56%-58% A. m. carica and 42%-44% A. m. macedonica. Spatial analysis showed the existence of a north-west to south-east dine in genetic differentiation. The bees in the north-west resemble A. m. carnica, while the bees in the south-east of the country are more similar to A. m. macedonica. Thus, the extent of A. m. macedonica within Serbia was greater than previously estimated. We define a line of hybridisation between A. m. carnica and A. m. macedonica within our study area. The dine of differentiation was still evident using a combination of genetic and spatial information, in spite of beekeeping activities including transhumance and breeding efforts.

Deleted in bladder cancer 1 (DBC1) is a tumour suppressor which is involved in the regulation of cell growth and programmed cell death. In this study we report the cloning and characterization of porcine DBC1 cDNA. RT-PCR cloning produced a cDNA with an open reading frame of 2,283 bp encoding a polypeptide of 761 amino acids with a predicted molecular mass of 88.6 kDa and estimated isoelectric point of 9.1. The encoded pig DBC1 protein shows a very high amino acid similarity to human (99 %) and to mouse (98 %) DBC1. The porcine DBC1 gene was mapped to chromosome 1. The nucleotide sequence of the promoter displayed a high degree of conservation of elements responsible for neuron-specific expression. The porcine DBC1 gene was found to be highly expressed in brain tissues. The methylation status of the porcine DBC1 gene was examined in brain and liver by bisulfite sequencing. Methylation percentages of 53-61 were observed for the gene body whereas significantly lower values (1-4 %) were found in exon 1 and the promoter sequence of DBC1. The sequences of the porcine DBC1 cDNA and the DBC1 promoter and exon 1 sequence have been submitted to DDBJ/EMBL/GenBank under the accession numbers KF733442 and KJ396193, respectively.

Aim. To study genes regulating the extracellular matrix (ECM) and investigate the tissue remodelling following liver resection in porcine. Methods. Four pigs with 60% partial hepatectomy- (PHx-) induced liver regeneration were studied over six weeks. Four pigs underwent sham surgery and another four pigs were used as controls of the normal liver growth. Liver biopsies were taken upon laparotomy, after three and six weeks. Gene expression profiles were obtained using porcine-specific oligonucleotide microarrays. Immunohistochemical staining was performed and a proliferative index was assessed. Results. More differentially expressed genes were associated with the regulation of ECM in the resection group compared to the sham and control groups. Secreted protein acidic and rich in cysteine (SPARC) and collagen 1, alpha 2 (COL1A2) were both upregulated in the early phase of liver regeneration, validated by immunopositive cells during the remodelling phase of liver regeneration. A broadened connective tissue was demonstrated by Masson’s Trichrome staining, and an immunohistochemical staining against pan-Cytokeratin (pan-CK) demonstrated a distinct pattern of migrating cells, followed by proliferating cell nuclear antigen (PCNA) positive nuclei. Conclusions. The present study demonstrates both a distinct pattern of PCNA positive nuclei and a deposition of ECM proteins in the remodelling phase of liver regeneration.