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Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet (Setaria viridis), a model species for studies of C4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet (Setaria italica). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a ‘loss of shattering’ trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR–Cas9 genome editing, we validated Less Shattering1 (SvLes1) as a gene whose product controls seed shattering. In S. italica, this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.Sequencing wild relatives of millet identifies genes that regulate yield and harvesting traits.

Main conclusion Misexpression of the AtNPC1-1 and AtNPC1-2 genes leads to altered sphingolipid metabolism, growth impairment, and male reproductive defects in a hemizygous Arabidopsis thaliana (L.) double-mutant population. Abolishing the expression of both gene copies has lethal effects. Abstract Niemann-Pick disease type C1 is a lysosomal storage disorder caused by mutations in the NPC1 gene. At the cellular level, the disorder is characterized by the accumulation of storage lipids and lipid trafficking defects. The Arabidopsis thaliana genome contains two genes (At1g42470 and At4g38350) with weak homology to mammalian NPC1. The corresponding proteins have 11 predicted membrane-spanning regions and contain a putative sterol-sensing domain. The At1g42470 protein is localized to the plasma membrane, while At4g38350 protein has a dual localization in the plasma and tonoplast membranes. A phenotypic analysis of T-DNA insertion mutants indicated that At1g42470 and At4g38350 (designated AtNPC1-1 and AtNPC1-2, respectively) have partially redundant functions and are essential for plant reproductive viability and development. Homozygous plants impaired in the expression of both genes were not recoverable. Plants of a hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 population were severely dwarfed and exhibited male gametophytic defects. These gene disruptions did not have an effect on sterol concentrations; however, hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 mutants had increased fatty acid amounts. Among these, fatty acid α-hydroxytetracosanoic acid (h24:0) occurs in plant sphingolipids. Follow-up analyses confirmed the accumulation of significantly increased levels of sphingolipids (assayed as hydrolyzed sphingoid base component) in the hemizygous double-mutant population. Certain effects of NPC1 misexpression may be common across divergent lineages of eukaryotes (sphingolipid accumulation), while other defects (sterol accumulation) may occur only in certain groups of eukaryotic organisms.

Abstract Mechanistic modeling indicates that stomatal conductance could be reduced to improve water use efficiency (WUE) in C4 crops. Genetic variation in stomatal density and canopy temperature was evaluated in the model C4 genus, Setaria. Recombinant inbred lines (RIL) derived from a Setaria italica x Setaria viridis cross were grown with ample or limiting water supply under field conditions in Illinois. An optical profilometer was used to rapidly assess stomatal patterning and canopy temperature was measured using infrared imaging. Stomatal density and canopy temperature were positively correlated but both were negatively correlated with total above-ground biomass. These trait relationships suggest a likely interaction between stomatal density and the other drivers of water use such as stomatal size and aperture. Multiple QTLs were identified for stomatal density and canopy temperature, including co-located QTLs on chromosomes 5 and 9. The direction of the additive effect of these QTLs on chromosome 5 and 9 were in accordance with the positive phenotypic relationship between these two traits. This suggests a common genetic architecture between stomatal patterning in the greenhouse and canopy transpiration in the field, while highlighting the potential of setaria as a model to understand the physiology and genetics of WUE in C4 species. Highlight This article reports a phenotypic and genetic relationship between two water use related traits operating at leaf level and canopy level in a C4 model crop species. Footnotes * Parthiban Thathapalli Prakash – tpparthiban{at}gmail.com, Darshi Banan – banan.darshi{at}gmail.com, Rachel E. Paul – repaul9{at}gmail.com, Maximilian J. Feldman – Max.Feldman{at}usda.gov, Ivan Baxter – IBaxter{at}danforthcenter.org, Dan Xie – xie243{at}purdue.edu, Luke Freyfogle – lukefreyfogle14{at}gmail.com, Andrew D.B. Leakey – leakey{at}illinois.edu

The biosynthesis of sterol molecules is both ubiquitous and essential in the eukaryotic lineage. Despite our advanced knowledge of the structural genes and enzymes involved in phytosterol biosynthesis in Arabidopsis thaliana, our understanding of how this biosynthetic pathway is regulated remains unsatisfactory. This is in stark contrast with to the case in other eukaryotes, where the mechanistic details of pathway regulation are well documented. In the context of this dissertation I utilized a data mining strategy to identify candidate regulatory proteins of sterol biosynthesis and examine their function using reverse genetics. The first study is a detailed characterization of bHLH93, a basic helix-loop-helix protein (bHLH) protein that was identified as a candidate transcriptional regulator of phytosterol metabolism based on the correlation of gene expression vectors in leaf/shoot tissue and the statistical enrichment of DNA motifs in the promoters of sterol biosynthetic genes. bHLH93 is a transcription factor that is localized to the nucleus in plant cells. bhlh93 mutants exhibit pleiotropic phenotypic defects including decreases in bolt length, downward curling of rosette leaves, and accumulation of dark green pigments when grown under continuous, moderately intense light. Metabolically, bhlh93 mutants exhibit dramatic alterations within isoprenoid metabolism (decreases in phytosterols and increases in plastidial isopreniods) and significant perturbations in lipid metabolism. Gene expression changes observed in bhlh93-1/bhlh61-1 double knockouts and a bHLH93 over-expression line suggest that bHLH93 is essential for the light-dependent coordination of development and metabolism in Arabidopsis thaliana. In a second study, I investigated the function of Arabidopsis proteins that contain a putative sterol-sensing domain (SSD). The Arabidopsis genome encodes two highly similar proteins with putative SSDs, AtSSD1 and AtSSD2. Based on sequence comparisons it appears that SSD proteins of plants are most similar to Niemann Pick Type C proteins found in other organisms. Our reverse genetic analysis suggests that AtSSD1 and AtSSD2 are at least partially redundant and essential for both reproductive and vegetative development. Analyses of AtSSD1/atssd1/atssd2/atssd2 double mutant lines suggest that these proteins are likely important in the regulation of fatty acid but not phytosterol metabolism.

We present a platinum-quality genome assembly for the model grass Setaria viridis, and high quality genomic sequences of 600+ wild accessions (average 42.6x coverage). Presence-absence variation (PAV) and single-nucleotide polymorphisms (SNPs) identify several subpopulations in North America. Using genome-wide association mapping plus CRISPR-Cas9 technology, we identified and validated Less Shattering1 (SvLES1), a gene for seed shattering with a retrotransposon insertion in the domesticated S. italica (foxtail millet) allele. We also identified a candidate gene for erect leaves, orthologous to the maize gene liguleless2. These results demonstrate the utility of the model plant S. viridis for complex trait dissection in panicoid crops.

The identifiable victim effect describes the stronger tendency to help a specific victim than to help a group of unidentified statistical victims. Our reanalysis of a meta-analysis on the effect by Lee and Freely (2016) using robust Bayesian meta-analysis suggested publication bias in the literature and the need to revisit the phenomenon. We conducted a pre-registered far replication and extension of Studies 1 and 3 in Small et al. (2007), a seminal demonstration of the identifiable victim effect, with hypothetical donations. We examined the impact of deliberative thinking on the identifiable victim effect both by directly informing participants of the effect (Study 1) and by providing an identified victim with statistical information (Study 3). We found no empirical support for the identifiable victim effect (ηp2 = .000, 95% CI [.000, .003]) and subsequently no support for debiasing such a phenomenon (ηp2 = .001, 95% CI[.000, .012]). These findings suggest that the identifiable victim may be better framed in terms of ‘scope-insensitivity’. In other words, rather than providing more to a single identified victim, participants seem to be insensitive to the number of victims affected. However, our study involved only hypothetical donations rather than a real-effort real-donation paradigm as in Small et al. (2007). Therefore, we hope that our results spark motivation for future high-powered replications with real money donations, ideally carried out as registered reports and in collaboration with proponents of the original effect. Materials, data, and code were made available on the OSF: https://osf.io/n4jkh/ .

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT–LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT–qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program. SARS-CoV-2 detection with LAMP-Seq combines isothermal amplification and barcoding with high-throughput sequencing.

Abstract Neovascular age-related macular degeneration (AMD) is a progressive eye disease and is a leading cause of vision loss in the Western world. Vascular endothelial growth factor inhibitors have become a mainstay of treatment for this disease. Currently, treatment options include three originator biologics with approvals for neovascular AMD (aflibercept, ranibizumab, and brolucizumab-dbll) and one biologic that is commonly used off-label for the condition (bevacizumab). In the USA, Medicare spending on these drugs consistently surpassed $4 billion per year between 2015 and 2019, driven by high prices and varying off-label use of bevacizumab, which is substantially cheaper than the other biologics used to treat neovascular AMD. In this article, we discuss how legal reform can improve market competition for biologic drugs, using AMD therapies as a case study. We chose this group of drugs for their significant contribution to Medicare spending, the price difference between approved therapies and intravitreal bevacizumab, and because there currently exists a large biosimilar pipeline with many drug candidates in the final stage of development. We propose mechanisms for anticipating and facilitating the market introduction of biosimilars, as well as changes to the pricing model in Medicare that can promote use of cost-effective therapies. Reforms such as empowering Medicare to negotiate drug prices may help ensure that introduction of new biologics and biosimilars for AMD will lower spending and increase patient access.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing COVID-19 outbreaks. Here, we present LAMP-Seq, a modified, highly scalable reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules/µl at 95% confidence, and near-perfect specificity for SARS-CoV-2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with Ct values up to 33, and a specificity of 99.7%, at material cost of 2.73 USD per sample. With a time-to-result less than 24 hours, low cost, and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.