No abstract

While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly 1 . The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE) 2 . Here we report the whole-genome sequencing and assembly of the desiccationtolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.The genomes of Arabidopsis 3 , rice 4 , poplar, grape and Sorghum 5 were first sequenced using high-quality and reiterative Sanger-based approaches producing a series of 'gold standard' reference genomes. The advent of next-generation sequencing (NGS) technologies reduced costs of sequencing substantially, which has enabled sequencing of over 100 plant genomes 1 . The quality of plant genome assemblies depends on genome size, ploidy, heterozygosity and sequence coverage, but most NGS-based genomes have on the order of tens of thousands of short contigs distributed in thousands of scaffolds. The short read lengths of NGS, inherent biases and non-random sequencing errors have resulted in highly fragmented draft genome assemblies that are not complete, which means they are missing biologically meaningful sequences including entire genes, regulatory regions, transposable elements, centromeres, telomeres and haplotype-specific structural variations. It is becoming clear from ENCODE projects that complete genomes are needed to better understand the importance of the non-coding regions of genomes 2 .More than 40% of calories consumed by humans are derived from grasses, and the grass family (Poaceae) is arguably the most important plant family with regard to global food security 6 . The size and complexity of most grass genomes has challenged progress in gene discovery and comparative genomics, although draft genomes are now available for most agriculturally important grasses 1 . The largest genome assemblies, such as maize (2,300 megabases (Mb)) 7 , barley (5,100 Mb) 8 and wheat (hexaploid, 1...

Tomatoes are a principal dietary source of carotenoids and flavonoids, both of which are highly beneficial for human health. Overexpression of genes encoding biosynthetic enzymes or transcription factors have resulted in tomatoes with improved carotenoid or flavonoid content, but never with both. We attempted to increase tomato fruit nutritional value by suppressing an endogenous photomorphogenesis regulatory gene, DET1, using fruit-specific promoters combined with RNA interference (RNAi) technology. Molecular analysis indicated that DET1 transcripts were indeed specifically degraded in transgenic fruits. Both carotenoid and flavonoid contents were increased significantly, whereas other parameters of fruit quality were largely unchanged. These results demonstrate that manipulation of a plant regulatory gene can simultaneously influence the production of several phytonutrients generated from independent biosynthetic pathways, and provide a novel example of the use of organ-specific gene silencing to improve the nutritional value of plant-derived products.

Small RNAs trigger repressive DNA methylation at thousands of transposable elements in a process called RNA-directed DNA methylation (RdDM). The molecular mechanism of RdDM is well characterized in Arabidopsis, yet the biological function remains unclear, as loss of RdDM in Arabidopsis causes no overt defects, even after generations of inbreeding. It is known that 24 nucleotide Pol IV-dependent siRNAs, the hallmark of RdDM, are abundant in flowers and developing seeds, indicating that RdDM might be important during reproduction. Here we show that, unlike Arabidopsis, mutations in the Pol IV-dependent small RNA pathway cause severe and specific reproductive defects in Brassica rapa. High rates of abortion occur when seeds have RdDM mutant mothers, but not when they have mutant fathers. Although abortion occurs after fertilization, RdDM function is required in maternal somatic tissue, not in the female gametophyte or the developing zygote, suggesting that siRNAs from the maternal soma might function in filial tissues. We propose that recently outbreeding species such as B. rapa are key to understanding the role of RdDM during plant reproduction.

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). InBrassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression duringB. rapaseed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wildtype and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T 2 statistic). Functional analyses of T 2 -selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.

SummaryThe tomato HIGH PIGMENT-2 gene encodes an orthologue of the Arabidopsis nuclear protein DE-ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light-regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper-responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post-transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper-responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3¢-terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential.