Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

A chromosome conformation capture ordered sequence of the barley genome.

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.

A novel allele of ASY3 is associated with greater meiotic stability in autotetraploid Arabidopsis lyrata.

In this study we performed a genotype-phenotype association analysis of meiotic stability in 10 autotetraploid Arabidopsis lyrata and A. lyrata/A. arenosa hybrid populations collected from the Wachau region and East Austrian Forealps. The aim was to determine the effect of eight meiosis genes under extreme selection upon adaptation to whole genome duplication. Individual plants were genotyped by high-throughput sequencing of the eight meiosis genes (ASY1, ASY3, PDS5b, PRD3, REC8, SMC3, ZYP1a/b) implicated in synaptonemal complex formation and phenotyped by assessing meiotic metaphase I chromosome configurations. Our results reveal that meiotic stability varied greatly (20-100%) between individual tetraploid plants and associated with segregation of a novel ASYNAPSIS3 (ASY3) allele derived from A. lyrata. The ASY3 allele that associates with meiotic stability possesses a putative in-frame tandem duplication (TD) of a serine-rich region upstream of the coiled-coil domain that appears to have arisen at sites of DNA microhomology. The frequency of multivalents observed in plants homozygous for the ASY3 TD haplotype was significantly lower than in plants heterozygous for ASY3 TD/ND (non-duplicated) haplotypes. The chiasma distribution was significantly altered in the stable plants compared to the unstable plants with a shift from proximal and interstitial to predominantly distal locations. The number of HEI10 foci at pachytene that mark class I crossovers was significantly reduced in a plant homozygous for ASY3 TD compared to a plant heterozygous for ASY3 ND/TD. Fifty-eight alleles of the 8 meiosis genes were identified from the 10 populations analysed, demonstrating dynamic population variability at these loci. Widespread chimerism between alleles originating from A. lyrata/A. arenosa and diploid/tetraploids indicates that this group of rapidly evolving genes may provide precise adaptive control over meiotic recombination in the tetraploids, the very process that gave rise to them.

Genomic erosion in a demographically recovered bird species during conservation rescue.

The pink pigeon (Nesoenas mayeri) is an endemic species of Mauritius that has made a remarkable recovery after a severe population bottleneck in the 1970s to early 1990s. Prior to this bottleneck, an ex situ population was established from which captive-bred individuals were released into free-living subpopulations to increase population size and genetic variation. This conservation rescue led to rapid population recovery to 400-480 individuals, and the species was twice downlisted on the International Union for the Conservation of Nature (IUCN) Red List. We analyzed the impacts of the bottleneck and genetic rescue on neutral genetic variation during and after population recovery (1993-2008) with restriction site-associated sequencing, microsatellite analyses, and quantitative genetic analysis of studbook data of 1112 birds from zoos in Europe and the United States. We used computer simulations to study the predicted changes in genetic variation and population viability from the past into the future. Genetic variation declined rapidly, despite the population rebound, and the effective population size was approximately an order of magnitude smaller than census size. The species carried a high genetic load of circa 15 lethal equivalents for longevity. Our computer simulations predicted continued inbreeding will likely result in increased expression of deleterious mutations (i.e., a high realized load) and severe inbreeding depression. Without continued conservation actions, it is likely that the pink pigeon will go extinct in the wild within 100 years. Conservation rescue of the pink pigeon has been instrumental in the recovery of the free-living population. However, further genetic rescue with captive-bred birds from zoos is required to recover lost variation, reduce expression of harmful deleterious variation, and prevent extinction. The use of genomics and modeling data can inform IUCN assessments of the viability and extinction risk of species, and it helps in assessments of the conservation dependency of populations.

La paloma rosada (Nesoenas mayeri) es una especie endémica de Mauricio que se ha recuperado impresionantemente después de un grave cuello de botella poblacional a principios de la década de 1970 que duró hasta inicios de la década de 1990. Antes de este cuello de botella se había establecido una población ex situ de la cual se liberaban individuos reproducidos en cautiverio a las subpoblaciones en libertad para incrementar la variación genética y el tamaño poblacional. Este rescate de conservación derivó en una recuperación rápida de la población (400-480 individuos) y la especie cambió positivamente de categoría dos veces en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN). Analizamos los impactos del cuello de botella y el rescate genético sobre la variación genética neutral durante y después de la recuperación poblacional (de 1993 a 2008) mediante secuenciación RAD, análisis de microsatélites y análisis genéticos cuantitativos de los datos del libro genealógico de 1112 aves ubicadas en zoológicos de Europa y los Estados Unidos. Usamos simulaciones por computadora para estudiar los cambios pronosticados en la variación genética y en la viabilidad poblacional del pasado hacia el futuro. La variación genética declinó rápidamente, a pesar de la recuperación poblacional, y el tamaño efectivo de la población fue aproximadamente un orden de magnitud más pequeño que el tamaño del censo. La especie contó con una carga genética elevada de casi 15 equivalentes letales para la longevidad. Nuestras simulaciones pronostican que la endogamia continua probablemente resultará en un incremento en la expresión de mutaciones deletéreas (es decir, una carga realizada elevada) y en una depresión endogámica severa. Sin acciones continuas para la conservación, es probable que la paloma rosada esté extinta en vida libre dentro de cien años. El rescate de conservación de la paloma rosada ha sido fundamental en la recuperación de la población silvestre; sin embargo, se requiere de un rescate genético adicional con las aves de reproducción en cautiverio de los zoológicos para recuperar la variación perdida, reducir la expresión de la variación deletérea dañina y prevenir la extinción. El uso de la genómica y los datos modelados puede orientar las valoraciones de la UICN sobre la viabilidad y el riesgo de extinción de las especies, además de que ayuda en la evaluación de la dependencia que tienen las poblaciones de la conservación.

Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. METHODS: Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. RESULTS: Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. CONCLUSIONS: Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

NanoOK: multi-reference alignment analysis of nanopore sequencing data, quality and error profiles.

MOTIVATION: The Oxford Nanopore MinION sequencer, currently in pre-release testing through the MinION Access Programme (MAP), promises long reads in real-time from an inexpensive, compact, USB device. Tools have been released to extract FASTA/Q from the MinION base calling output and to provide basic yield statistics. However, no single tool yet exists to provide comprehensive alignment-based quality control and error profile analysis--something that is extremely important given the speed with which the platform is evolving. RESULTS: NanoOK generates detailed tabular and graphical output plus an in-depth multi-page PDF report including error profile, quality and yield data. NanoOK is multi-reference, enabling detailed analysis of metagenomic or multiplexed samples. Four popular Nanopore aligners are supported and it is easily extensible to include others. AVAILABILITY AND IMPLEMENTATION: NanoOK is an open-source software, implemented in Java with supporting R scripts. It has been tested on Linux and Mac OS X and can be downloaded from https://github.com/TGAC/NanoOK. A VirtualBox VM containing all dependencies and the DH10B read set used in this article is available from http://opendata.tgac.ac.uk/nanook/. A Docker image is also available from Docker Hub--see program documentation https://documentation.tgac.ac.uk/display/NANOOK. CONTACT: richard.leggett@tgac.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A family of diatom-like silicon transporters in the siliceous loricate choanoflagellates.

Biosilicification is widespread across the eukaryotes and requires concentration of silicon in intracellular vesicles. Knowledge of the molecular mechanisms underlying this process remains limited, with unrelated silicon-transporting proteins found in the eukaryotic clades previously studied. Here, we report the identification of silicon transporter (SIT)-type genes from the siliceous loricate choanoflagellates Stephanoeca diplocostata and Diaphanoeca grandis. Until now, the SIT gene family has been identified only in diatoms and other siliceous stramenopiles, which are distantly related to choanoflagellates among the eukaryotes. This is the first evidence of similarity between SITs from different eukaryotic supergroups. Phylogenetic analysis indicates that choanoflagellate and stramenopile SITs form distinct monophyletic groups. The absence of putative SIT genes in any other eukaryotic groups, including non-siliceous choanoflagellates, leads us to propose that SIT genes underwent a lateral gene transfer event between stramenopiles and loricate choanoflagellates. We suggest that the incorporation of a foreign SIT gene into the stramenopile or choanoflagellate genome resulted in a major metabolic change: the acquisition of biomineralized silica structures. This hypothesis implies that biosilicification has evolved multiple times independently in the eukaryotes, and paves the way for a better understanding of the biochemical basis of silicon transport through identification of conserved sequence motifs.

Tumor suppressor p16INK4a determines sensitivity of human cells to transformation by cooperating cellular oncogenes.

The Ink4a/Arf locus encodes two distinct proteins, both of which may contribute to senescence and tumor suppression. We find that human diploid fibroblasts (HDFs) that are specifically deficient for p16INK4a achieve anchorage independence when transduced with retroviruses encoding telomerase (hTERT) and either Ras or Myc. Significantly, Ras and Myc together enable the cells to form tumors in nude mice but at a frequency that suggests additional genetic changes. All five tumors analyzed expressed high levels of Ras and retained functional p53, although two showed downregulation of Arf. Cytogenetic analyses identified clonal chromosomal alterations that may have contributed to tumorigenesis, but the tumor cells were essentially diploid.

Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples.

Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms. To test its potential for enrichment of rarer species within metagenomic samples, we create a synthetic mock community and construct sequencing libraries with a range of mean read lengths. Enrichment is up to 13.87-fold for the least abundant species in the longest read length library; factoring in reduced yields from rejecting molecules the calculated efficiency raises this to 4.93-fold. Finally, we introduce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlate strongly with mock and complex real-world microbial communities.

Genome sequence of the vertebrate gut symbiont Lactobacillus reuteri ATCC 53608.

Lactobacillus reuteri, inhabiting the gastrointestinal tracts of a range of vertebrates, is a true symbiont with effects established as beneficial to the host. Here we describe the draft genome of L. reuteri ATCC 53608, isolated from a pig. The genome sequence provides important insights into the evolutionary changes underlying host specialization.

Draft genome sequence of Streptomyces strain S4, a symbiont of the leaf-cutting ant Acromyrmex octospinosus.

Streptomyces spp. are common symbionts of the leaf-cutting ant species Acromyrmex octospinosus, which feeds on basidiomycete fungus leaf matter and harvests the lipid- and carbohydrate-rich gongylidia as a food source. A. octospinosus and other ant genera use antifungal compounds produced by Streptomyces spp. and other actinomycetes in order to help defend their fungal gardens from parasitic fungi. Herein, we report the draft genome sequence of Streptomyces strain S4, an antifungal-producing symbiont of A. octospinosus.

Complete genome sequence of the proteolytic Clostridium botulinum type A5 (B3') strain H04402 065.

H04402 065 is one of a very small group of strains of proteolytic Clostridium botulinum that form type A5 neurotoxin. Here, we report the complete 3.9-Mb genome sequence and annotation of strain H04402 065, which was isolated from a botulism patient in the United Kingdom in 2004.

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus.

BACKGROUND: Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive. RESULTS: In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus. CONCLUSIONS: Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes.

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations.

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates of Cryptosporidium species.

Clinical and environmental isolates of pathogens are often unique and may be unculturable, yielding a very limited amount of DNA for genetic studies. Cryptosporidium in particular are difficult to propagate. Whole genome amplification (WGA) is a valuable technique for amplifying genomic material. In this study, we tested 5 WGA commercial kits using Cryptosporidium clinical isolates. DNA of 5 C. hominis and 5 C. parvum clinical isolates and C. parvum IOWA reference strain were used. The majority of the samples were amplified by all of the kits tested. The integrity and fidelity of the amplified genomic DNA were assessed by sequence analysis of several PCR products of varying length. We found evidence that one kit in particular may be more error prone while another seemed the more suitable kit for Cryptosporidium clinical samples, generating high molecular weight DNA from all the samples with high fidelity. Thus WGA was found to be a useful technique for producing amplified DNA suitable for downstream genotyping techniques and archiving of Cryptosporidium clinical isolates.

A low-cost pipeline for soil microbiome profiling.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low-cost genomic DNA extraction method for soil samples. We also present an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad-leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low-cost genomic DNA extraction method for soil samples that can be coupled to an Illumina-compatible simple two-step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high-quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large-scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.

A Genome Assembly of the Barley 'Transformation Reference' Cultivar Golden Promise.

Barley (Hordeum vulgare) is one of the most important crops worldwide and is also considered a research model for the large-genome small grain temperate cereals. Despite genomic resources improving all the time, they are limited for the cv Golden Promise, the most efficient genotype for genetic transformation. We have developed a barley cv Golden Promise reference assembly integrating Illumina paired-end reads, long mate-pair reads, Dovetail Chicago in vitro proximity ligation libraries and chromosome conformation capture sequencing (Hi-C) libraries into a contiguous reference assembly. The assembled genome of 7 chromosomes and 4.13Gb in size, has a super-scaffold N50 after Chicago libraries of 4.14Mb and contains only 2.2% gaps. Using BUSCO (benchmarking universal single copy orthologous genes) as evaluation the genome assembly contains 95.2% of complete and single copy genes from the plant database. A high-quality Golden Promise reference assembly will be useful and utilized by the whole barley research community but will prove particularly useful for CRISPR-Cas9 experiments.