Population Structure and Diversity in European Honey Bees (Apismellifera L.)-An Empirical Comparison of Pool and Individual Whole-Genome Sequencing.

BACKGROUND: Whole-genome sequencing has become routine for population genetic studies. Sequencing of individuals provides maximal data but is rather expensive and fewer samples can be studied. In contrast, sequencing a pool of samples (pool-seq) can provide sufficient data, while presenting less of an economic challenge. Few studies have compared the two approaches to infer population genetic structure and diversity in real datasets. Here, we apply individual sequencing (ind-seq) and pool-seq to the study of Western honey bees (Apis mellifera). METHODS: We collected honey bee workers that belonged to 14 populations, including 13 subspecies, totaling 1347 colonies, who were individually (139 individuals) and pool-sequenced (14 pools). We compared allele frequencies, genetic diversity estimates, and population structure as inferred by the two approaches. RESULTS: Pool-seq and ind-seq revealed near identical population structure and genetic diversities, albeit at different costs. While pool-seq provides genome-wide polymorphism data at considerably lower costs, ind-seq can provide additional information, including the identification of population substructures, hybridization, or individual outliers. CONCLUSIONS: If costs are not the limiting factor, we recommend using ind-seq, as population genetic structure can be inferred similarly well, with the advantage gained from individual genetic information. Not least, it also significantly reduces the effort required for the collection of numerous samples and their further processing in the laboratory.

Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

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.

Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoform α4.

Na+/K+-ATPase is responsible for maintaining electrochemical gradients of Na+ and K+, which is essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase is composed of four different polypeptides (α1-α4) encoded by different genes. Na,K-ATPase α4, encoded by the ATP1A4 gene, is expressed in testis and in male germ cells of humans, rats and mice. The α4 polypeptide has an important role in sperm motility, and is essential for male fertility. Here we present the RT-PCR cloning and characterization of the porcine ATP1A4 cDNA coding for Na⁺/K⁺-ATPase polypeptide α4. The Na⁺/K⁺-ATPase polypeptide α4, consisting of 1030 amino acids, displays a high homology with its human counterpart (86%). Phylogenetic analysis demonstrated that porcine Na⁺/K⁺-ATPase polypeptide α4 is closely related to other mammalian counterparts. In addition, the genomic structure of the porcine ATP1A4 gene was determined, and the intron-exon organization was found to be similar to that of the human ATP1A4 gene. The promoter sequence for the porcine ATP1A4 gene was also identified. Investigation of the genetic variation in the porcine ATP1A4 gene revealed a missense A/G SNP in exon 18. This A/G polymorphism results in a substitution of a methionine to a glycine residue (M888G). A very high overall DNA methylation rate of the ATP1A4 gene, 70-80%, was observed in both brain and liver. Expression analysis demonstrated that the porcine ATP1A4 gene is predominantly expressed in testis. The sequence of the porcine ATP1A4 cDNA encoding the Na⁺/K⁺-ATPase α4 protein has been submitted to GenBank under the accession number GenBank Accession No. MG587082.

A-to-I RNA editing of the IGFBP7 transcript increases during aging in porcine brain tissues.

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.

Differential A-to-I RNA editing of the serotonin-2C receptor G-protein-coupled, HTR2C, in porcine brain tissues.

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.

Molecular characterization and analysis of the porcine NURR1 gene.

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.

Cloning and characterization of the porcine DBC1 gene encoding deleted in bladder cancer.

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.

Porcine SLITRK1: Molecular cloning and characterization.

The membrane protein SLITRK1 functions as a developmentally regulated stimulator of neurite outgrowth and variants in this gene have been implicated in Tourette syndrome. In the current study we have cloned and characterized the porcine SLITRK1 gene. The genomic organization of SLITRK1 lacks introns, as does its human and mouse counterparts. RT-PCR cloning revealed two SLITRK1 transcripts: a full-length mRNA and a transcript variant that results in a truncated protein. The encoded SLITRK1 protein, consisting of 695 amino acids, displays a very high homology to human SLITRK1 (99%). The porcine SLITRK1 gene is expressed exclusively in brain tissues.

Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Porcine synapsin 1: SYN1 gene analysis and functional characterization of the promoter.

Synapsin 1 (SYN1) is a phosphoprotein involved in nerve signal transmission. The porcine SYN1 promoter orthologue was cloned and characterized to provide a means of expressing a transgene specifically in neurons. The nucleotide sequence of the promoter displayed a high degree of conservation of elements responsible for neuron-specific expression. Expression analysis of SYN1 demonstrated presence of transcript during embryonic development. Analysis of GFP expression in transgenic zebrafish embryos suggests that the pig SYN1 promoter directs expression in neuronal cells. Thus, the SYN1 promoter is a good candidate for use in the generation of pig models of human neurodegenerative disorders.

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level.

The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the "electron transport chain" and neuronal differentiation, emphasizing that "tissue important" genes are regulated at several levels. Furthermore, our analysis shows that the "across tissue approach" has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.