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April 21, 2020  |  

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR. © 2019 American Society of Plant Biologists. All rights reserved.

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April 21, 2020  |  

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Fast and accurate genomic analyses using genome graphs.

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, thus impairing analysis accuracy. Here we present a graph reference genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million insertions and deletions (indels). The pipeline processes one whole-genome sequencing sample in 6.5?h using a system with 36?CPU cores. We show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, with unaffected specificity. Structural variations incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is an important advance toward fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

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April 21, 2020  |  

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae. © 2019 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Genome Sequence of Jaltomata Addresses Rapid Reproductive Trait Evolution and Enhances Comparative Genomics in the Hyper-Diverse Solanaceae.

Within the economically important plant family Solanaceae, Jaltomata is a rapidly evolving genus that has extensive diversity in flower size and shape, as well as fruit and nectar color, among its ~80 species. Here, we report the whole-genome sequencing, assembly, and annotation, of one representative species (Jaltomata sinuosa) from this genus. Combining PacBio long reads (25×) and Illumina short reads (148×) achieved an assembly of ~1.45?Gb, spanning ~96% of the estimated genome. Ninety-six percent of curated single-copy orthologs in plants were detected in the assembly, supporting a high level of completeness of the genome. Similar to other Solanaceous species, repetitive elements made up a large fraction (~80%) of the genome, with the most recently active element, Gypsy, expanding across the genome in the last 1-2 Myr. Computational gene prediction, in conjunction with a merged transcriptome data set from 11 tissues, identified 34,725 protein-coding genes. Comparative phylogenetic analyses with six other sequenced Solanaceae species determined that Jaltomata is most likely sister to Solanum, although a large fraction of gene trees supported a conflicting bipartition consistent with substantial introgression between Jaltomata and Capsicum after these species split. We also identified gene family dynamics specific to Jaltomata, including expansion of gene families potentially involved in novel reproductive trait development, and loss of gene families that accompanied the loss of self-incompatibility. This high-quality genome will facilitate studies of phenotypic diversification in this rapidly radiating group and provide a new point of comparison for broader analyses of genomic evolution across the Solanaceae.

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April 21, 2020  |  

Heterochromatin-enriched assemblies reveal the sequence and organization of the Drosophila melanogaster Y chromosome.

Heterochromatic regions of the genome are repeat-rich and poor in protein coding genes, and are therefore underrepresented in even the best genome assemblies. One of the most difficult regions of the genome to assemble are sex-limited chromosomes. The Drosophila melanogaster Y chromosome is entirely heterochromatic, yet has wide-ranging effects on male fertility, fitness, and genome-wide gene expression. The genetic basis of this phenotypic variation is difficult to study, in part because we do not know the detailed organization of the Y chromosome. To study Y chromosome organization in D. melanogaster, we develop an assembly strategy involving the in silico enrichment of heterochromatic long single-molecule reads and use these reads to create targeted de novo assemblies of heterochromatic sequences. We assigned contigs to the Y chromosome using Illumina reads to identify male-specific sequences. Our pipeline extends the D. melanogaster reference genome by 11.9 Mb, closes 43.8% of the gaps, and improves overall contiguity. The addition of 10.6 MB of Y-linked sequence permitted us to study the organization of repeats and genes along the Y chromosome. We detected a high rate of duplication to the pericentric regions of the Y chromosome from other regions in the genome. Most of these duplicated genes exist in multiple copies. We detail the evolutionary history of one sex-linked gene family, crystal-Stellate While the Y chromosome does not undergo crossing over, we observed high gene conversion rates within and between members of the crystal-Stellate gene family, Su(Ste), and PCKR, compared to genome-wide estimates. Our results suggest that gene conversion and gene duplication play an important role in the evolution of Y-linked genes. Copyright © 2019 Chang and Larracuente.

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April 21, 2020  |  

MSC: a metagenomic sequence classification algorithm.

Metagenomics is the study of genetic materials directly sampled from natural habitats. It has the potential to reveal previously hidden diversity of microscopic life largely due to the existence of highly parallel and low-cost next-generation sequencing technology. Conventional approaches align metagenomic reads onto known reference genomes to identify microbes in the sample. Since such a collection of reference genomes is very large, the approach often needs high-end computing machines with large memory which is not often available to researchers. Alternative approaches follow an alignment-free methodology where the presence of a microbe is predicted using the information about the unique k-mers present in the microbial genomes. However, such approaches suffer from high false positives due to trading off the value of k with the computational resources. In this article, we propose a highly efficient metagenomic sequence classification (MSC) algorithm that is a hybrid of both approaches. Instead of aligning reads to the full genomes, MSC aligns reads onto a set of carefully chosen, shorter and highly discriminating model sequences built from the unique k-mers of each of the reference sequences.Microbiome researchers are generally interested in two objectives of a taxonomic classifier: (i) to detect prevalence, i.e. the taxa present in a sample, and (ii) to estimate their relative abundances. MSC is primarily designed to detect prevalence and experimental results show that MSC is indeed a more effective and efficient algorithm compared to the other state-of-the-art algorithms in terms of accuracy, memory and runtime. Moreover, MSC outputs an approximate estimate of the abundances.The implementations are freely available for non-commercial purposes. They can be downloaded from https://drive.google.com/open?id=1XirkAamkQ3ltWvI1W1igYQFusp9DHtVl. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020  |  

Genetic characterization and potential molecular dissemination mechanism of tet(31) gene in Aeromonas caviae from an oxytetracycline wastewater treatment system.

Recently, the rarely reported tet(31) tetracycline resistance determinant was commonly found in Aeromonas salmonicida, Gallibacterium anatis, and Oblitimonas alkaliphila isolated from farming animals and related environment. However, its distribution in other bacteria and potential molecular dissemination mechanism in environment are still unknown. The purpose of this study was to investigate the potential mechanism underlying dissemination of tet(31) by analysing the tet(31)-carrying fragments in A. caviae strains isolated from an aerobic biofilm reactor treating oxytetracycline bearing wastewater. Twenty-three A. caviae strains were screened for the tet(31) gene by polymerase chain reaction (PCR). Three strains (two harbouring tet(31), one not) were subjected to whole genome sequencing using the PacBio RSII platform. Seventeen A. caviae strains carried the tet(31) gene and exhibited high resistance levels to oxytetracycline with minimum inhibitory concentrations (MICs) ranging from 256 to 512?mg/L. tet(31) was comprised of the transposon Tn6432 on the chromosome of A. caviae, and Tn6432 was also found in 15 additional tet(31)-positive A. caviae isolates by PCR. More important, Tn6432 was located on an integrative conjugative element (ICE)-like element, which could mediate the dissemination of the tet(31)-carrying transposon Tn6432 between bacteria. Comparative analysis demonstrated that Tn6432 homologs with the structure ISCR2-?phzF-tetR(31)-tet(31)-?glmM-sul2 were also carried by A. salmonicida, G. anatis, and O. alkaliphila, suggesting that this transposon can be transferred between species and even genera. This work provides the first report on the identification of the tet(31) gene in A. caviae, and will be helpful in exploring the dissemination mechanisms of tet(31) in water environment.Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020  |  

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

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April 21, 2020  |  

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast.

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ~20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti.

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, Brugia timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti (Wb). Here we use population genomics to reconstruct the probable route and timing of migration of Wb strains that currently infect Africa, Haiti, and Papua New Guinea (PNG). We used selective whole genome amplification to sequence 42 whole genomes of single Wb worms from populations in Haiti, Mali, Kenya, and PNG. Our results are consistent with a hypothesis of an Island Southeast Asia or East Asian origin of Wb. Our demographic models support divergence times that correlate with the migration of human populations. We hypothesize that PNG was infected at two separate times, first by the Melanesians and later by the migrating Austronesians. The migrating Austronesians also likely introduced Wb to Madagascar where later migrations spread it to continental Africa. From Africa, Wb spread to the New World during the transatlantic slave trade. Genome scans identified 17 genes that were highly differentiated among Wb populations. Among these are genes associated with human immune suppression, insecticide sensitivity, and proposed drug targets. Identifying the distribution of genetic diversity in Wb populations and selection forces acting on the genome will build a foundation to test future hypotheses and help predict response to current eradication efforts. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020  |  

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30°?N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.

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April 21, 2020  |  

Computational aspects underlying genome to phenome analysis in plants.

Recent advances in genomics technologies have greatly accelerated the progress in both fundamental plant science and applied breeding research. Concurrently, high-throughput plant phenotyping is becoming widely adopted in the plant community, promising to alleviate the phenotypic bottleneck. While these technological breakthroughs are significantly accelerating quantitative trait locus (QTL) and causal gene identification, challenges to enable even more sophisticated analyses remain. In particular, care needs to be taken to standardize, describe and conduct experiments robustly while relying on plant physiology expertise. In this article, we review the state of the art regarding genome assembly and the future potential of pangenomics in plant research. We also describe the necessity of standardizing and describing phenotypic studies using the Minimum Information About a Plant Phenotyping Experiment (MIAPPE) standard to enable the reuse and integration of phenotypic data. In addition, we show how deep phenotypic data might yield novel trait-trait correlations and review how to link phenotypic data to genomic data. Finally, we provide perspectives on the golden future of machine learning and their potential in linking phenotypes to genomic features. © 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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