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Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid ( Triticum turgidum , AABB) and hexaploid ( Triticum aestivum , AABBDD) wheat 1 , 2 . Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping 4 , 5 . We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147  T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement. The genome sequence of Triticum urartu , the progenitor of the A subgenome of hexaploid wheat, provides insight into genome duplication during grass evolution.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley ( Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion. The International Barley Genome Sequencing Consortium reports sequencing and assembly of a reference genome for barley, Hordeum vulgare . Barley genome sequenced Triticeae grasses, which include barley, wheat and rye, are widely cultivated plants with particularly complex genomes and evolutionary histories. Sequencing of the barley genome has been particularly challenging owing to its large size and particular genomic features, such as an abundance of repetitive elements. Nils Stein and colleagues of the International Barley Genome Sequencing Consortium report sequencing and assembly of a reference genome for barley ( Hordeumvulgare L). They use a combined approach of hierarchical shotgun sequencing of bacterial artificial chromosomes, genome mapping on nanochannel arrays and chromosome-scale scaffolding with Hi-C sequencing. This brings the first comprehensive, completely ordered assembly of the pericentromeric regions of a Triticeae genome. The authors also sequenced and examined genetic diversity in the exomes of 96 European elite barley lines with a spring or winter growth habit, and highlight the utility of this resource for cereal genomics and breeding programs.

Sugarcane ( Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum , involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence. Sugarcane ( Saccharum spp.) is a crop of major economic significance but has complex genome structure. Here, the authors generate a BAC-based monoploid sugarcane reference sequence.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function 1 – 3 . To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)—a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)—a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers 1 , 4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome—a key intermediate in its total synthesis 1 —from five episomes in 10  days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly 5 , 6 , along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections 1 , 7 , 8 , we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months. BAC stepwise insertion synthesis (BASIS) can be used to build synthetic genomes for diverse organisms, and continuous genome synthesis (CGS) enables the rapid synthesis of entire Escherichia coli genomes from functional designs.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

An artificial chromosome-like plasmid was developed for Corynebacterium glutamicum.The C. glutamicum artificial chromosome facilitates stepwise genome replacements of ~50 kb.In total, 361 kb of synthetic DNA was integrated into the C. glutamicum genome (11%). Recent advances in genome synthesis have relied on scalable DNA assembly and delivery, and efficient recombination techniques. While these methods have enabled rapid progress for Escherichia coli and yeast, they are often inadequate for other microorganisms. Here, we devised a Corynebacterium glutamicum artificial chromosome (CAC), which combines a replicating system from a closely related strain with an innate partitioning system. This CAC vector can efficiently deliver DNA fragments up to 56 kb and maintain stability in C. glutamicum. Leveraging the CAC vector, we developed CAC Excision Enhanced Recombination (CACEXER), a streamlined strategy for iterative genome replacements in C. glutamicum. Using this approach, we integrated 361 kb (11%) of synthetic DNA into the genome, creating semi-synCG-A. This strain paves the way to establish C. glutamicum as the third industrial microorganism, alongside E. coli and Saccharomyces cerevisiae, to undergo large-scale genome synthesis. [Display omitted] This work establishes a bacterial artificial chromosome (BAC)-like vector for Corynebacterium glutamicum and introduces the streamlined C. glutamicum Artificial Chromosome (CAC) Excision Enhanced Recombination (CACEXER) strategy for de novo genome synthesis of this industrially important Gram-positive microbe. To date, we have successfully integrated 361 kb (or 11%) of synthetic DNA into the genome. To further enhance this CAC-based approach and accelerate genome synthesis, we aim to incorporate recent advancements in large-scale DNA assembly and delivery. In addition, genome debugging to mitigate growth defects will be crucial for achieving a fully synthetic C. glutamicum genome. Based on our current progress, we propose that this technology has reached a Technology Readiness Level (TRL) between 3 and 4, as defined by NASA. We developed an artificial chromosome plasmid (CAC) for large-scale genome replacement of Corynebacterium glutamicum, accelerating the genome synthesis for this organism. This work offers design principles for advancing de novo genome design and synthesis for industrially relevant Gram-positive microbes.

The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo . This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19. Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo . This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

De novo assembly and phasing of the genome of an individual from Korea using a combination of different sequencing approaches provides a useful population-specific reference genome and represents the most contiguous human genome assembly so far. A Korean human genome Jeong-Sun Seo and colleagues report de novo assembly and phasing of the genome of an individual from Korea using a combination of PacBio long-read sequencing, Illumina short-read sequencing, 10X Genomics linked reads, bacterial artificial chromosome (BAC) sequencing and BioNano Genomics optical mapping. This provides a useful population-specific reference genome and represents the most contiguous human genome assembly to date. The authors use this to close gaps in the human reference genome and map structural variation. Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1 ) using single-molecule real-time sequencing 2 , next-generation mapping 3 , microfluidics-based linked reads 4 , and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6 . This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Background The number of de novo genome sequence assemblies is increasing exponentially; however, relatively few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of conserved non-coding elements is associated with the positions of evolutionary breakpoint regions. Results We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest. Conclusions The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80% assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements, especially interchromosomal, are fixed during evolution without deleterious effects.

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site–leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.