Menu
September 22, 2019

The N6-adenine methylation in yeast genome profiled by single-molecule technology.

The most common and abundant DNA modification is 5-meth- ylcytosine (5mC), which has been well-established as an epigenetic mark regulating gene expression in eukaryotes (Jones, 2012). Another DNA modification N6-methyldeoxyadenosine (6mA), pre- viously reported as a widespread DNA methylation in prokaryotes, plays an important role in gene expression, DNA replication, DNA repair, cell cycle progression and host-pathogen interaction (Messer and Noyer-Weidner, 1988; Lu et al., 1994; Collier et al., 2007). The knowledge of 6mA in eukaryotes has been very limited until the recent development of high-throughput sequencing and high-sensitive mass spectrometry technologies, which have greatly contributed to the investigation of 6mA in fungi, animals and plants (Fu et al., 2015; Greer et al., 2015; Zhang et al., 2015; Koziol et al., 2016; Liu et al., 2016; Wu et al., 2016; Liang et al., 2017; Mondo et al., 2017). Recent studies revealed that 6mA abundance is vari- able, and it is relative higher in Chlamydomonas and early- diverging fungi species than other eukaryotes. The distribution pat- terns of 6mA and their functions are not quite conserved among or- ganisms. 6mA was found enriched near the transcription start sites (TSS) in Chlamydomonas (Fu et al., 2015) and at the repeats in Drosophila, Mus musculus and Danio rerio (Zhang et al., 2015; Liu et al., 2016; Wu et al., 2016), and commonly depleted from gene exons in Xenopus laevis and M. musculus (Koziol et al., 2016). In several species, 6mA was associated with transcriptionally active genes (Fu et al., 2015; Mondo et al., 2017), and it was also found correlated with gene silencing in mammalian embryonic stem cells (Wu et al., 2016).


September 22, 2019

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.


September 22, 2019

DNA N6-adenine methylation in Arabidopsis thaliana.

DNA methylation on N6-adenine (6mA) has recently been found to be a potentially epigenetic mark in several unicellular and multicellular eukaryotes. However, its distribution patterns and potential functions in land plants, which are primary producers for most ecosystems, remain largely unknown. Here we report global profiling of 6mA sites at single-nucleotide resolution in the genome of Arabidopsis thaliana at different developmental stages using single-molecule real-time sequencing. 6mA sites are widely distributed across the Arabidopsis genome and enriched over the pericentromeric heterochromatin regions. 6mA occurs more frequently in gene bodies than intergenic regions. Analysis of 6mA methylomes and RNA sequencing data demonstrates that 6mA frequency positively correlates with the gene expression level and the transition from vegetative to reproductive growth in Arabidopsis. Our results uncover 6mA as a DNA mark associated with actively expressed genes in Arabidopsis, suggesting that 6mA serves as a hitherto unknown epigenetic mark in land plants. Copyright © 2018 Elsevier Inc. All rights reserved.


September 22, 2019

Comparative genomics of cocci-shaped Sporosarcina strains with diverse spatial isolation.

Cocci-shaped Sporosarcina strains are currently one of the few known cocci-shaped spore-forming bacteria, yet we know very little about the genomics. The goal of this study is to utilize comparative genomics to investigate the diversity of cocci-shaped Sporosarcina strains that differ in their geographical isolation and show different nutritional requirements.For this study, we sequenced 28 genomes of cocci-shaped Sporosarcina strains isolated from 13 different locations around the world. We generated the first six complete genomes and methylomes utilizing PacBio sequencing, and an additional 22 draft genomes using Illumina sequencing. Genomic analysis revealed that cocci-shaped Sporosarcina strains contained an average genome of 3.3 Mb comprised of 3222 CDS, 54 tRNAs and 6 rRNAs, while only two strains contained plasmids. The cocci-shaped Sporosarcina genome on average contained 2.3 prophages and 15.6 IS elements, while methylome analysis supported the diversity of these strains as only one of 31 methylation motifs were shared under identical growth conditions. Analysis with a 90% identity cut-off revealed 221 core genes or ~?7% of the genome, while a 30% identity cut-off generated a pan-genome of 8610 genes. The phylogenetic relationship of the cocci-shaped Sporosarcina strains based on either core genes, accessory genes or spore-related genes consistently resulted in the 29 strains being divided into eight clades.This study begins to unravel the phylogenetic relationship of cocci-shaped Sporosarcina strains, and the comparative genomics of these strains supports identification of several new species.


September 22, 2019

The evolution of genomic and epigenomic features in two Pleurotus fungi.

Pleurotus tuoliensis (Bailinggu, designated Pt) and P. eryngii var. eryngii (Xingbaogu, designated Pe) are highly valued edible mushrooms. We report de novo assemblies of high-quality genomes for both mushrooms based on PacBio RS II sequencing and annotation of all identified genes. A comparative genomics analysis between Pt and Pe with P. ostreatus as an outgroup taxon revealed extensive genomic divergence between the two mushroom genomes primarily due to the rapid gain of taxon-specific genes and disruption of synteny in either taxon. The re-appraised phylogenetic relationship between Pt and Pe at the genome-wide level validates earlier proposals to designate Pt as an independent species. Variation of the identified wood-decay-related gene content can largely explain the variable adaptation and host specificity of the two mushrooms. On the basis of the two assembled genome sequences, methylomes and the regulatory roles of DNA methylation in gene expression were characterized and compared. The genome, methylome and transcriptome data of these two important mushrooms will provide valuable information for advancing our understanding of the evolution of Pleurotus and related genera and for facilitating genome- and epigenome-based strategies for mushroom breeding.


September 22, 2019

A whole genome assembly of the horn fly, Haematobia irritans, and prediction of genes with roles in metabolism and sex determination.

Haematobia irritans, commonly known as the horn fly, is a globally distributed blood-feeding pest of cattle that is responsible for significant economic losses to cattle producers. Chemical insecticides are the primary means for controlling this pest but problems with insecticide resistance have become common in the horn fly. To provide a foundation for identification of genomic loci for insecticide resistance and for discovery of new control technology, we report the sequencing, assembly, and annotation of the horn fly genome. The assembled genome is 1.14 Gb, comprising 76,616 scaffolds with N50 scaffold length of 23 Kb. Using RNA-Seq data, we have predicted 34,413 gene models of which 19,185 have been assigned functional annotations. Comparative genomics analysis with the Dipteran flies Musca domestica L., Drosophila melanogaster, and Lucilia cuprina, show that the horn fly is most closely related to M. domestica, sharing 8,748 orthologous clusters followed by D. melanogaster and L. cuprina, sharing 7,582 and 7,490 orthologous clusters respectively. We also identified a gene locus for the sodium channel protein in which mutations have been previously reported that confers target site resistance to the most common class of pesticides used in fly control. Additionally, we identified 276 genomic loci encoding members of metabolic enzyme gene families such as cytochrome P450s, esterases and glutathione S-transferases, and several genes orthologous to sex determination pathway genes in other Dipteran species. Copyright © 2018 Konganti et al.


September 22, 2019

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for Rvi12 (Vb)-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.


September 22, 2019

Sea cucumber genome provides insights into saponin biosynthesis and aestivation regulation.

Echinoderms exhibit several fascinating evolutionary innovations that are rarely seen in the animal kingdom, but how these animals attained such features is not well understood. Here we report the sequencing and analysis of the genome and extensive transcriptomes of the sea cucumber Apostichopus japonicus, a species from a special echinoderm group with extraordinary potential for saponin synthesis, aestivation and organ regeneration. The sea cucumber does not possess a reorganized Hox cluster as previously assumed for all echinoderms, and the spatial expression of Hox7 and Hox11/13b potentially guides the embryo-to-larva axial transformation. Contrary to the typical production of lanosterol in animal cholesterol synthesis, the oxidosqualene cyclase of sea cucumber produces parkeol for saponin synthesis and has “plant-like” motifs suggestive of convergent evolution. The transcriptional factors Klf2 and Egr1 are identified as key regulators of aestivation, probably exerting their effects through a clock gene-controlled process. Intestinal hypometabolism during aestivation is driven by the DNA hypermethylation of various metabolic gene pathways, whereas the transcriptional network of intestine regeneration involves diverse signaling pathways, including Wnt, Hippo and FGF. Decoding the sea cucumber genome provides a new avenue for an in-depth understanding of the extraordinary features of sea cucumbers and other echinoderms.


September 22, 2019

Genome-wide DNA methylation and transcriptome changes in Mycobacterium tuberculosis with rifampicin and isoniazid resistance

We investigated the genome-wide DNA methylation and transcriptome changes in M. tuberculosis with rifampicin or isoniazid resistance. Single-molecule real-time (SMRT) sequencing and microarray technology were performed to expound DNA methylation profiles and differentially expressed genes in rifampicin or isoniazid resis- tant M. tuberculosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis and meth- ylated regulatory network analysis were conducted by online forecasting databases. Integrated analysis of DNA methylation and transcriptome revealed that 335 differentially methylated genes (175 hypermethylated and 160 hypomethylated) and 132 significant differentially expressed genes (68 up-regulated and 63 down-regulated) were found to be regulated by both rifampicin and isoniazid in M. tuberculosis H37Rv. Correlation analysis showed that differential methylated genes were negatively correlated with their transcriptional levels in rifampicin or isoniazid resistant strains. KEGG pathway analysis indicated that nitrogen metabolism pathway is closely related to differ- entially methylated genes induced by rifampicin and isoniazid. KEGG also suggested that differentially expressed genes in rifampicin or isoniazid-resistant strains may play different roles in regulating signal transduction events. Furthermore, five differentially methylated candidate genes (Rv0840c, Rv2243, Rv0644c, Rv2386c and Rv1130) in rifampicin resistant strains and three genes (Rv0405, Rv0252 and Rv0908) in isoniazid-resistant strains were verified the existence of protein-protein interaction in STRING database. Integrated DNA methylation and transcrip- tome analyses provide an epigenetic overview of rifampicin and isoniazid-induced antibiotic resistance in M. tuber- culosis H37Rv. Several interesting genes and regulatory pathways may provide valuable resources for epigenetic studies in M. tuberculosis antibiotic resistance.


September 22, 2019

Recurrent loss, horizontal transfer, and the obscure origins of mitochondrial introns in diatoms (Bacillariophyta).

We sequenced mitochondrial genomes from five diverse diatoms (Toxarium undulatum, Psammoneis japonica, Eunotia naegelii, Cylindrotheca closterium, and Nitzschia sp.), chosen to fill important phylogenetic gaps and help us characterize broadscale patterns of mitochondrial genome evolution in diatoms. Although gene content was strongly conserved, intron content varied widely across species. The vast majority of introns were of group II type and were located in the cox1 or rnl genes. Although recurrent intron loss appears to be the principal underlying cause of the sporadic distributions of mitochondrial introns across diatoms, phylogenetic analyses showed that intron distributions superficially consistent with a recurrent-loss model were sometimes more complicated, implicating horizontal transfer as a likely mechanism of intron acquisition as well. It was not clear, however, whether diatoms were the donors or recipients of horizontally transferred introns, highlighting a general challenge in resolving the evolutionary histories of many diatom mitochondrial introns. Although some of these histories may become clearer as more genomes are sampled, high rates of intron loss suggest that the origins of many diatom mitochondrial introns are likely to remain unclear.


September 22, 2019

Mosaic structure as the main feature of Mycobacterium bovis BCG genomes

Background: The genome stability of attenuated live BCG vaccine preventing the acute forms of childhood tuberculosis is an important aspect of vaccine production. The pur- pose of our study was a whole genome comparative analysis of BCG sub-strains and identification of potential triggers of sub-strains’ transition. Results: Genomes of three BCG Russia seed lots (1963, 1982, 2006 years) have been sequenced, and the stability of vaccine sub-strain genomes has been confirmed. A com- parative genome analysis of nine Mycobacterium bovis BCG and three M. bovis strains revealed their specific genome features associated with prophage profiles. A number of prophage-coded homologs to Caudovirales ORFs were common to all BCG genomes. Prophage profiles of BCG Tice and BCG Montreal genomes were unique and coded homologs to herpes viruses ORFs. The data of phylogenetic analysis of BCG sub-strain groups based on whole genome sequences and genome restriction maps were in con- gruence with prophage profiles. The only fragmentary similarity of specific prophage sequences of BCG Tice, BCG Montreal, and BCG Russia 368 in pair-wise alignments was observed, suggesting the impact of prophages on mosaic structure of genomes. Conclusions: The whole genome sequencing approach is essential for genomes with mosaic structure, harboring numerous prophage sequences. Tools for prophage search are effective instruments in this analysis.


September 22, 2019

Fungal Epigenomics: Detection and Analysis.

Across Eukaryota, DNA modifications play an important role in regulation of gene expression. While 5-methylcytosine (5mC) has been explored in depth, other modifications such as 6-methyladenine (6 mA) have historically been overlooked, in part due to technical difficulties in collecting/analyzing these data. However, recent technological advances have enabled exploration of these marks with much greater detail and on a larger scale. In this chapter, we discuss multiple methods for identifying and analyzing both 5mC and 6 mA across fungi.


September 22, 2019

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.© 2018 Zhu et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.


September 22, 2019

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X).The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions.We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.