White and other fur colourations and hybridization in golden jackals (Canis aureus) in the Carpathian basin.

The golden jackal (Canis aureus) is a reoccurring species in the centre of the Carpathian basin, in Hungary. In total, 31 golden jackal tissue samples were collected, from 8 white-coated, 2 black-coated and one mottled animal across Hungary. Sequences and fragment length polymorphisms were studied for white colour (MC1R), and for black coat colouration (CBD103). In each white animal, the most widespread mutation causing white fur colour in dogs in homozygous form was detected. Three animals were found to carry the mutation in heterozygous form. The two black golden jackals were heterozygous for the 3 bp deletion in CBD103 that mutation for black coat colouration in dogs, and one of them also carried the mutation causing white fur. None of the white animals showed signs of hybridization, but both the black and the mottled coloured individuals were found to be hybrids based on genetic testing. Kinship was found three times, twice between white animals, and once between a white animal and an agouti animal carrying the mutation of white coat. Our results confirm the findings that golden jackal-dog hybrids may occur without human intervention, and the detected mutation causing white fur colour in golden jackals could possibly be due to an early hybridization event.

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy.

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis.

During muscle cell differentiation, the alternatively spliced, acidic ß-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the ß-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid ß-domain variants, as well as by a disordered ß-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the ß-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that ß-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process.

Extensive proteome and functional genomic profiling of variability between genetically identical human B-lymphoblastoid cells.

In life-science research isogenic B-lymphoblastoid cell lines (LCLs) are widely known and preferred for their genetic stability - they are often used for studying mutations for example, where genetic stability is crucial. We have shown previously that phenotypic variability can be observed in isogenic B-lymphoblastoid cell lines. Isogenic LCLs present well-defined phenotypic differences on various levels, for example on the gene expression level or the chromatin level. Based on our investigations, the phenotypic variability of the isogenic LCLs is accompanied by certain genetic variation too. We have developed a compendium of LCL datasets that present the phenotypic and genetic variability of five isogenic LCLs from a multiomic perspective. In this paper, we present additional datasets generated with Next Generation Sequencing techniques to provide genomic and transcriptomic profiles (WGS, RNA-seq, single cell RNA-seq), protein-DNA interactions (ChIP-seq), together with mass spectrometry and flow cytometry datasets to monitor the changes in the proteome. We are sharing these datasets with the scientific community according to the FAIR principles for further investigations.

An Ultra-Rare Manifestation of an X-Linked Recessive Disorder: Duchenne Muscular Dystrophy in a Female Patient.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle dystrophy. Patients are characterized by muscle weakness, gross motor delay, and elevated serum creatinine kinase (CK) levels. The disease is caused by mutations in the DMD gene located on the X chromosome. Due to the X-linked recessive inheritance pattern, DMD most commonly affects males, who are generally diagnosed between the age of 3-5 years. Here we present an ultra-rare manifestation of DMD in a female patient. Cytogenetic examination showed that she has a t(X;10)(p21.1;p12.1) translocation, which turned out to affect the DMD gene with one of the breakpoints located in exon 54 (detected by genome sequencing). The X-inactivation test revealed skewed X-inactivation (ratio 99:1). Muscle histology and dystrophin immunohistochemistry showed severe dystrophic changes and highly reduced dystrophin expression, respectively. These results, in accordance with the clinical picture and a highly elevated serum CK, led to the diagnosis of DMD. In conclusion, although in very rare cases, DMD can manifest in female patients as well. In this case, a balanced X-autosome reciprocal translocation disrupts the DMD gene and skewed X-inactivation leads to the manifestation of the DMD phenotype.

Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers.

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.

Mapping and DNA sequence characterisation of the Rysto locus conferring extreme virus resistance to potato cultivar 'White Lady'.

Virus resistance genes carried by wild plant species are valuable resources for plant breeding. The Rysto gene, conferring a broad spectrum of durable resistance, originated from Solanum stoloniferum and was introgressed into several commercial potato cultivars, including 'White Lady', by classical breeding. Rysto was mapped to chromosome XII in potato, and markers used for marker-assisted selection in breeding programmes were identified. Nevertheless, there was no information on the identity of the Rysto gene. To begin to reveal the identification of Rysto, fine-scale genetic mapping was performed which, in combination with chromosome walking, narrowed down the locus of the gene to approximately 1 Mb. DNA sequence analysis of the locus identified six full-length NBS-LRR-type (short NLR-type) putative resistance genes. Two of them, designated TMV2 and TMV3, were similar to a TMV resistance gene isolated from tobacco and to Y-1, which co-segregates with Ryadg, the extreme virus resistance gene originated from Solanum andigena and localised to chromosome XI. Furthermore, TMV2 of 'White Lady' was found to be 95% identical at the genomic sequence level with the recently isolated Rysto gene of the potato cultivar 'Alicja'. In addition to the markers identified earlier, this work generated five tightly linked new markers which can serve potato breeding efforts for extreme virus resistance.

ChIPSummitDB: a ChIP-seq-based database of human transcription factor binding sites and the topological arrangements of the proteins bound to them.

ChIP-seq reveals genomic regions where proteins, e.g. transcription factors (TFs) interact with DNA. A substantial fraction of these regions, however, do not contain the cognate binding site for the TF of interest. This phenomenon might be explained by protein-protein interactions and co-precipitation of interacting gene regulatory elements. We uniformly processed 3727 human ChIP-seq data sets and determined the cistrome of 292 TFs, as well as the distances between the TF binding motif centers and the ChIP-seq peak summits. ChIPSummitDB enables the analysis of ChIP-seq data using multiple approaches. The 292 cistromes and corresponding ChIP-seq peak sets can be browsed in GenomeView. Overlapping SNPs can be inspected in dbSNPView. Most importantly, the MotifView and PairShiftView pages show the average distance between motif centers and overlapping ChIP-seq peak summits and distance distributions thereof, respectively. In addition to providing a comprehensive human TF binding site collection, the ChIPSummitDB database and web interface allows for the examination of the topological arrangement of TF complexes genome-wide. ChIPSummitDB is freely accessible at http://summit.med.unideb.hu/summitdb/. The database will be regularly updated and extended with the newly available human and mouse ChIP-seq data sets.

Correction: Expansion of Capsicum annum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

[This corrects the article DOI: 10.1371/journal.pone.0200207.].

Expansion of Capsicum annuum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important transcriptional and post-transcriptional regulators controlling a wide variety of physiological processes including fruit development. Data are, however, limited for their potential roles in developmental processes determining economically important traits of crops. The current study aimed to discover and characterize differentially expressed miRNAs and siRNAs in sweet pepper (Capsicum annuum) during fruit expansion. High-throughput sequencing was employed to determine the small regulatory RNA expression profiles in various fruit tissues, such as placenta, seed, and flesh at 28 and 40 days after anthesis. Comparative differential expression analyses of conserved, already described and our newly predicted pepper-specific miRNAs revealed that fruit expansion is accompanied by an increasing level of miRNA-mediated regulation of gene expression. Accordingly, ARGONAUTE1 protein, the primary executor of miRNA-mediated regulation, continuously accumulated to an extremely high level in the flesh. We also identified numerous pepper-specific, heterochromatin-associated 24-nt siRNAs (hetsiRNAs) which were extremely abundant in the seeds, as well as 21-nt and 24-nt phased siRNAs (phasiRNAs) that were expressed mainly in the placenta and the seeds. This work provides comprehensive tissue-specific miRNA and siRNA expression landscape for a developing pepper fruit. We identified several novel, abundantly expressing tissue- and pepper-specific small regulatory RNA species. Our data show that fruit expansion is associated with extensive changes in sRNA abundance, raising the possibility that manipulation of sRNA pathways may be employed to improve the quality and quantity of the pepper fruit.

The red deer Cervus elaphus genome CerEla1.0: sequencing, annotating, genes, and chromosomes.

We present here the de novo genome assembly CerEla1.0 for the red deer, Cervus elaphus, an emblematic member of the natural megafauna of the Northern Hemisphere. Humans spread the species in the South. Today, the red deer is also a farm-bred animal and is becoming a model animal in biomedical and population studies. Stag DNA was sequenced at 74× coverage by Illumina technology. The ALLPATHS-LG assembly of the reads resulted in 34.7 × 103 scaffolds, 26.1 × 103 of which were utilized in Cer.Ela1.0. The assembly spans 3.4 Gbp. For building the red deer pseudochromosomes, a pre-established genetic map was used for main anchor points. A nearly complete co-linearity was found between the mapmarker sequences of the deer genetic map and the order and orientation of the orthologous sequences in the syntenic bovine regions. Syntenies were also conserved at the in-scaffold level. The cM distances corresponded to 1.34 Mbp uniformly along the deer genome. Chromosomal rearrangements between deer and cattle were demonstrated. 2.8 × 106 SNPs, 365 × 103 indels and 19368 protein-coding genes were identified in CerEla1.0, along with positions for centromerons. CerEla1.0 demonstrates the utilization of dual references, i.e., when a target genome (here C. elaphus) already has a pre-established genetic map, and is combined with the well-established whole genome sequence of a closely related species (here Bos taurus). Genome-wide association studies (GWAS) that CerEla1.0 (NCBI, MKHE00000000) could serve for are discussed.

Draft Genome Sequence of an Atypical Highly Virulent Rabbit Staphylococcus aureus Strain.

Rabbit staphylococcosis is one of the most important diseases in industrial rabbit production. We report here the draft genome sequence of Staphylococcus aureus strain 380/11, an atypical highly virulent (aHV) rabbit Staphylococcus aureus strain.

The full mitochondrial genomes of Mangalica pig breeds and their possible origin.

The mitogenomes of one animal of each of the three Mangalica breeds, Blonde, Red, and Swallow-belly were assembled from reads obtained by Next Generation Sequencing of the three genomes. Features of the mitogenomes were identical in the three breeds, apart from a second tRNA-Val gene on the L strand in Swallow-belly. Phylogenetic comparison of the three mitogenomes with 112 full mtDNA sequences clearly put Mangalicas into the European clade. Comparing the mitogenome of eight Mangalica animals revealed particular differences between them. The mitogenome of some Mangalicas was closely related to the Croatian Turopolje breed and this indicates either the common origin of their maternal lineages or admixture of some populations of the breeds. However, the origin of the mitogenome of certain purebred Mangalicas kept in the Hungarian Mangalica Gene Reserve still remains unknown.

Genome Sequences of Multidrug-Resistant Salmonella enterica subsp. enterica Serovar Infantis Strains from Broiler Chicks in Hungary.

Three strains of Salmonella enterica serovar Infantis isolated from healthy broiler chickens from 2012 to 2013 have been sequenced. Comparison of these and previously published S Infantis genome sequences of broiler origin in 1996 and 2004 will provide new insight into the genome evolution and recent spread of S Infantis in poultry.

Motif oriented high-resolution analysis of ChIP-seq data reveals the topological order of CTCF and cohesin proteins on DNA.

BACKGROUND: ChIP-seq provides a wealth of information on the approximate location of DNA-binding proteins genome-wide. It is known that the targeted motifs in most cases can be found at the peak centers. A high resolution mapping of ChIP-seq peaks could in principle allow the fine mapping of the protein constituents within protein complexes, but the current ChIP-seq analysis pipelines do not target the basepair resolution strand specific mapping of peak summits. RESULTS: The approach proposed here is based on i) locating regions that are bound by a sufficient number of proteins constituting a complex; ii) determining the position of the underlying motif using either a direct or a de novo motif search approach; and iii) determining the exact location of the peak summits with respect to the binding motif in a strand specific manner. We applied this method for analyzing the CTCF/cohesin complex, which holds together DNA loops. The relative positions of the constituents of the complex were determined with one-basepair estimated accuracy. Mapping the positions on a 3D model of DNA made it possible to deduce the approximate local topology of the complex that allowed us to predict how the CTCF/cohesin complex locks the DNA loops. As the positioning of the proteins was not compatible with previous models of loop closure, we proposed a plausible "double embrace" model in which the DNA loop is held together by two adjacent cohesin rings in such a way that the ring anchored by CTCF to one DNA duplex encircles the other DNA double helix and vice versa. CONCLUSIONS: A motif-centered, strand specific analysis of ChIP-seq data improves the accuracy of determining peak positions. If a genome contains a large number of binding sites for a given protein complex, such as transcription factor heterodimers or transcription factor/cofactor complexes, the relative position of the constituent proteins on the DNA can be established with an accuracy that allow one to deduce the local topology of the protein complex. The proposed high resolution mapping approach of ChIP-seq data is applicable for detecting the contact topology of DNA-binding protein complexes.

[Letter to the Editor] Comparison of small RNA next-generation sequencing with and without isolation of small RNA fraction.

Address correspondence to Ferenc Marincs, Agricultural Biotechnology Institute, NARIC, Szent-Györgyi Albert u. 4., Gödöllö, Hungary. E-mail: marincs.ferenc@abc.naik.hu.

Complete mitochondrial genome sequence of a Hungarian red deer (Cervus elaphus hippelaphus) from high-throughput sequencing data and its phylogenetic position within the family Cervidae.

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 mitochondrial DNA analysis, which can be used to estimate phylogenetic relationships among animal taxa and for molecular phylogenetic evolution analysis. With the development of sequencing technology, more and more mitochondrial sequences have been made available in public databases, including whole mitochondrial DNA sequences. These data have been used for phylogenetic analysis of animal species, and for studies of evolutionary processes. We determined the complete mitochondrial genome of a Central European red deer, Cervus elaphus hippelaphus, from Hungary by a next generation sequencing technology. The mitochondrial genome is 16 354 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region, all of which are arranged similar as in other vertebrates. We made phylogenetic analyses with the new sequence and 76 available mitochondrial sequences of Cervidae, using Bos taurus mitochondrial sequence as outgroup. We used 'neighbor joining' and 'maximum likelihood' methods on whole mitochondrial genome sequences; the consensus phylogenetic trees supported monophyly of the family Cervidae; it was divided into two subfamilies, Cervinae and Capreolinae, and five tribes, Cervini, Muntiacini, Alceini, Odocoileini, and Capreolini. The evolutionary structure of the family Cervidae can be reconstructed by phylogenetic analysis based on whole mitochondrial genomes; which method could be used broadly in phylogenetic evolutionary analysis of animal taxa.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BACKGROUND: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. RESULTS: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. CONCLUSIONS: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

Draft Genome Sequence of a Highly Virulent Rabbit Staphylococcus aureus Strain.

We report the draft genome sequence of Staphylococcus aureus Sp17, a typical highly virulent (HV) rabbit strain. As current medicine apparently fails to effectively reduce disease and economical losses caused by this organism, it is essential to gain better insight on its genomic arrangement.