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July 7, 2019

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 7, 2019

DNA methylation profiling using long-read Single Molecule Real-Time bisulfite sequencing (SMRT-BS).

For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300-500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5-2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.


July 7, 2019

pSY153-MDR, a p12969-DIM-related mega plasmid carrying blaIMP-45 and armA, from clinical Pseudomonas putida.

This work characterized mega plasmid pSY153-MDR, carrying blaIMP-45 and armA, from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. blaIMP-45 and armA coexisted in a Tn1403-Tn1548 region, which was generated from homologous recombination of Tn1403- and Tn1548-like transposons. The second copy of armA was a component of the ISCR28-armA-?ISCR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.


July 7, 2019

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.© 2017 John Wiley & Sons Ltd.


July 7, 2019

Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite.

Successful process development for the bioleaching of mineral ores, particularly the refractory copper sulfide ore chalcopyrite, remains a challenge in regions where freshwater is scarce and source water contains high concentrations of chloride ion. In this study, a pure isolate of Acidihalobacter prosperus strain F5 was characterized for its ability to leach base metals from sulfide ores (pyrite, chalcopyrite and pentlandite) at increasing chloride ion concentrations. F5 successfully released base metals from ores including pyrite and pentlandite at up to 30gL(-1) chloride ion and chalcopyrite up to 18gL(-1) chloride ion. In order to understand the genetic mechanisms of tolerance to high acid, saline and heavy metal stress the genome of F5 was sequenced and analysed. As well as being the first strain of Ac. prosperus to be isolated from Australia it is also the first complete genome of the Ac. prosperus species to be sequenced. The F5 genome contains genes involved in the biosynthesis of compatible solutes and genes encoding monovalent cation/proton antiporters and heavy metal transporters which could explain its abilities to tolerate high salinity, acidity and heavy metal stress. Genome analysis also confirmed the presence of genes involved in copper tolerance. The study demonstrates the potential biotechnological applicability of Ac. prosperus strain F5 for saline water bioleaching of mineral ores. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

Complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea.

This study aimed to determine the complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea.Genomic DNA was sequenced using a PacBio RS II system. The obtained genome was annotated and antimicrobial resistance and virulence genes were identified.The sequenced genome possessed a mecA homologue (mecA1) that was almost identical to that of other oxacillin-susceptible S. sciuri strains, whereas the staphylococcal cassette chromosome mec (SCCmec) was not detected. Moreover, various antimicrobial resistance genes conferring resistance to ß-lactams, aminoglycosides, phenicols, tetracycline and macrolide-lincosamide-streptogramin B (MLSB) antimicrobials were identified.The SNUDS-18 genome and its associated genomic data will provide important insights into the biodiversity of the S. sciuri group as well as valuable information for the control of this potential pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.


July 7, 2019

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes.© The Authors 2017. Published by Oxford University Press.


July 7, 2019

Comparative analysis of the radish genome with Brassica genomes

Raphanus sativus L. includes an annual root vegetable crop, radish, and diverse wild species. R. sativus has a long history of domestication, but its phylogenetic position in the tribe Brassiceae is controversial. A comprehensive analysis of the R. sativus genome will provide fundamental information about the structure of its genome, evolutionary features of polyploidy, and significant insight for phylogenetic delimitation of this species. Diverse genomic resources, including a high-density genetic map, clone libraries, cytogenetic data, and transcriptome data, have been developed to sequence the genome. Recently, the R. sativus cv. ‘WK10039’ (2n = 18, 510.8 Mb) genome was sequenced and assembled into nine chromosome pseudomolecules spanning >98% of the gene space. Comparative mapping of the tPCK-like ancestral genome based on conserved ortholog set markers and proteome comparison revealed that the R. sativus genome has intermediate characteristics between the Brassica A/C and B genomes with triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between R. sativus and diploid Brassica species provide genomic evidence for species delimitation of R. sativus and reconstruction of the mesohexaploid ancestral genome.


July 7, 2019

Genomics of parallel adaptation at two timescales in Drosophila.

Two interesting unanswered questions are the extent to which both the broad patterns and genetic details of adaptive divergence are repeatable across species, and the timescales over which parallel adaptation may be observed. Drosophila melanogaster is a key model system for population and evolutionary genomics. Findings from genetics and genomics suggest that recent adaptation to latitudinal environmental variation (on the timescale of hundreds or thousands of years) associated with Out-of-Africa colonization plays an important role in maintaining biological variation in the species. Additionally, studies of interspecific differences between D. melanogaster and its sister species D. simulans have revealed that a substantial proportion of proteins and amino acid residues exhibit adaptive divergence on a roughly few million years long timescale. Here we use population genomic approaches to attack the problem of parallelism between D. melanogaster and a highly diverged conger, D. hydei, on two timescales. D. hydei, a member of the repleta group of Drosophila, is similar to D. melanogaster, in that it too appears to be a recently cosmopolitan species and recent colonizer of high latitude environments. We observed parallelism both for genes exhibiting latitudinal allele frequency differentiation within species and for genes exhibiting recurrent adaptive protein divergence between species. Greater parallelism was observed for long-term adaptive protein evolution and this parallelism includes not only the specific genes/proteins that exhibit adaptive evolution, but extends even to the magnitudes of the selective effects on interspecific protein differences. Thus, despite the roughly 50 million years of time separating D. melanogaster and D. hydei, and despite their considerably divergent biology, they exhibit substantial parallelism, suggesting the existence of a fundamental predictability of adaptive evolution in the genus.


July 7, 2019

Genetic maps and whole genome sequences of radish

Radish, Raphanus sativus L., is a member of Brassicaceae, to which Arabidopsis thaliana, a model plant in plant biology, belongs, as do other Brassica species including important crops. However, genetic and genomic studies of radish have been behind those of Arabidopsis and Brassica. In this decade, much effort has been made to develop genetic resources for radish, e.g., DNA markers, genetic maps, and whole genome sequences. Studies using the obtained information have revealed the genome structure of radish in terms of ancestral karyotype and have also prompted the identification of genes for agronomically important traits in radish through a map-based cloning strategy and quantitative trait locus analysis. In this chapter, we review the evolving development of radish genetic map in the past 15 years and the current status of genome sequencing of radish. We also introduce the latest strategy for the construction of a high-density genetic map using next-generation sequencing technology and propose a prospective direction of genetics and genomics research in radish which would be helpful for researchers and breeders in their efforts to promote radish breeding programs efficiently.


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