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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Root genetics in the field to understand drought adaptation and carbon sequestration (Final Scientific/Technical Report)

For all crop plants, roots play a critical role in growth. Roots anchor the plants, and are the primary site of nutrient and water uptake. Roots are also the main source of C to soil in the form of root tissues and exudates, and thus greatly influence SOM stocks. To perform these functions, primary roots extend into soil, producing a network of branching roots of characteristic form, known as its root system architecture (RSA). RSA varies among species, and among varieties within a species that are adapted to different environments. Root traits are major targets for the second green revolution because of their potential to improve crop productivity, increase drought tolerance and nutrient acquisition, and increase C capture of soil. Improving the quality of roots in maize will be particularly valuable, since this crop is planted on over 92 million acres annually in the US. The future sustainability of agricultural systems relies on their ability to enhance soil organic matter (SOM) storage and reduce GHG emissions, while maintaining or enhancing productivity. This program had two components, Sensors and Models. For the first component, we designed and built a high-throughput phenotyping platform for root pulling of maize plants. This eliminated the physical labor of manually pulling up plants and reduced the number of personnel required down to one. The standardized pulling mechanism allowed recording force curves during the pulling process, providing additional information. We validated that the maximum force for pulling the root system was well-correlated with the root system mass and provided root crowns for further RSA analysis. These root crowns identified significant correlations with 2D root area and root depth, along with 3D root volume, total root length and number of root tips. We then used this system for field-based studies in maize on the genetics of root system architecture and its relation to nitrogen-use efficiency (NUE), including using lines relevant to the Corteva breeding program. Varieties were also evaluated at Corteva sites in the cornbelt and Danforth farm in Missouri, to establish responses across sites. From these studies we have identified genetic loci associated with root traits and created mutant lines for these loci and correlations of root traits with NUE. For the Models component, we worked to incorporate root and soil characteristics into the MEMS 2.0 soil and ecosystem biogeochemical model. Existing soil C models, such as Century, are unable to represent specific root trait interactions with the soil environment and therefore to accurately forecast the potential C sequestration benefits of root breeding under different climatic and soil type conditions. We have developed the MEMS 2.0 ecosystem biogeochemical model to improve quantification of farm-scale soil carbon and greenhouse gas emissions. The new knowledge and large datasets produced by this project will be used to develop and drive an innovative model capable of forecasting the impacts on soil C stocks and nutrient dynamics. An innovation was to use the empirical data from the field studies (in 1, above) to model genetic variation in nitrogen use efficiencies and soil C input. Our work demonstrated that maize root-derived C rapidly replaces existing soil C and after 3 years of continuous maize, up to 20% of soil organic C in the topsoil (0-15cm) and 3% in the subsoil (15-30cm) was contributed by maize. However, this contribution did not entirely represent a net increase. Root C contribution to soil was affected by maize genetics. We have analyzed soils derived from the CSU field trials for C and N stocks, in the different soil physical fractions represented by the MEMS model, using both physical fractionation with elemental analyses, and Fourier transformed infrared spectroscopy. Data will be used to link crop nitrogen use efficiencies with soil C sequestration and provide data to bridge the field trials with the model development, for verification of model predictions. The project had a number of successful outcomes: we have used the new phenotyping platform to identify new genetic loci that can enhance root phenotypes; we have partnered with multiple maize seed companies phenotype varieties in their breeding programs; we have developed the MEMS model that can help inform industry on the potential for carbon sequestration in the agricultural sector, and which is now available at the CSU Soil Carbon Solutions Center for use.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Probing the limits of genetic recoding using multi-omics-guided evolution

Engineering the genetic code—by reassigning multiple of the 64 natural codons—enables making organisms resistant to all viruses, preventing genetic information exchange, and allowing the biosynthesis of genetically encoded unnatural polymers. However, synonymous codon replacement—recoding—is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using genome synthesis, directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple synthetic Escherichia coli genomes. We construct six partially recoded E. coli strains bearing up to 45.8% of a synthetic genome with a deleterious 57-codon genetic code. As our analyses revealed widespread defects—including unassigned codons in Syn61 and Syn57—we apply multi-omics to revise our genome design and mitigate defects. Using multi-omics, we show that recoding induces transcriptional and translational changes leading to fitness defects under hundreds of conditions. Finally, we develop a multi-omics-guided evolution strategy that rapidly restores fitness, enabling genome synthesis with radical changes.

Nyerges, Akos [Harvard Medical School, Boston, MA ↗
DOE OSTI2025

FluxRETAP: a REaction TArget Prioritization genome-scale modeling technique for selecting genetic targets

MOTIVATION: Metabolic engineering is rapidly evolving as a result of new advances in synthetic biology tools and automation platforms that enable high throughput strain construction, as well as the development of machine learning tools (ML) for biology. However, selecting genetic engineering targets that effectively guide the metabolic engineering process is still challenging. ML can provide predictive power for synthetic biology, but current technical limitations prevent the independent use of ML approaches without previous biological knowledge. RESULTS: Here, we present FluxRETAP, a simple and computationally inexpensive method that leverages the prior mechanistic knowledge embedded in genome-scale models for suggesting targets for genetic overexpression, downregulation or deletion, with the final goal of increasing the production of a desired metabolite. This method can provide a list of desirable engineering targets that can be combined with current ML pipelines. FluxRETAP captured 100% of reaction targets experimentally verified to improve Escherichia coli isoprenol production, 50% of targets that experimentally improved taxadiene production in E. coli and ∼60% of genetic targets from a verified minimal constrained cut-set in Pseudomonas putida, while providing additional high priority targets that could be tested. Overall, FluxRETAP is an efficient algorithm for identifying a prioritized list of testable genetic and reaction targets. AVAILABILITY AND IMPLEMENTATION: FluxRETAP is implemented in python and released under the creative commons license. The implementation and code are freely available at: https://github.com/JBEI/FluxRETAP.

Czajka, Jeffrey J↗
DOE OSTI2025

Evaluating genetic and environmental controls on silicon accumulation in Populus trichocarpa leaves

Background and aims: Silicon (Si) accumulation and formation of non-crystalline silica particles (phytoliths) inside plant cells contribute to plant function, soil carbon storage, and Si reservoirs in soils. A better understanding of how genetic and environmental factors control Si accumulation may improve understanding of Si cycling between plants and soil and its impacts on other ecosystem processes. Methods: This study examined how genetic potential for Si uptake and silicification translated to Si concentrations and phytolith formation in Populus trichocarpa leaves. Leaves were collected from four different genotypes of cultivated poplar trees that were grown in a common garden and possess established differences in leaf-level gene expression related to Si transport and silicification. Scanning electron microscopy and subsequent image analysis were used to quantify Si concentrations and phytolith abundance and features. Results: Phytoliths were identified in nearly all leaves but were often absent at very low (< 0.5 wt.%) Si concentrations at the leaf surface. The proportion of leaf area occupied by phytoliths increased as Si content increased. Silicon content and phytolith abundance at the leaf surface did not vary across genotypes, but surface Si was weakly correlated with plant available Si in soil. Conclusion: Phytoliths were prevalent in P. trichocarpa leaves; however, the genetic potential for Si uptake and silicification at the leaf-level poorly predicted foliar Si and phytolith abundance. Uniformly low expression of Si uptake genes in the roots may have reduced differences across genotypes. Consequently, Si accumulation in leaves was likely more strongly influenced by soil Si bioavailability, phenology, or additional environmental factors.

Image analysis↗
DOE OSTI2026

Genetic variations and their interaction with thirdhand smoke exposure on anxiety and memory in Collaborative Cross mice

Thirdhand smoke (THS) is linked to adverse health effects, but the effect of genetic variations on behavioral outcomes is poorly understood. To investigate this, we assessed anxiety- and memory-related behaviors in 820 mice from 21 strains of the genetically diverse Collaborative Cross (CC) mouse that were exposed to THS from 4 through 10 weeks of age. Anxiety was evaluated with a light/dark box assay with a previously established risk score system. Females were generally more sensitive: THS reduced anxiety risk in strains CC013, CC019, and CC051, but increased risk in CC036 and CC061, while males showed no significant effects. Memory was tested using passive avoidance: impairments were observed in both sexes in CC016 and CC019, with sex-dependent effects in CC002 and CC051. A genome-wide association study identified 2,347 SNPs associated with anxiety and 1,568 SNPs with memory, with 32 and 85 SNPs, respectively, interacting with THS exposure. Enrichment analyses revealed distinct biological processes underlying susceptibility, including axonogenesis, synapse organization, cognition, and learning and memory. KEGG pathway analysis identified distinct genetic pathways, including GTPase binding and GTPase regulatory activity, that act as critical molecular switches in the brain that regulate synaptic plasticity, dendritic spine structure, and neuronal signaling, directly influencing anxiety-like behaviors and memory formation. These findings show that THS exposure affects neurobehavioral outcomes in a sex- and genotype-dependent manner, highlighting critical gene-environment interactions and providing a foundation for mechanistic insights into THS neurotoxicity

Anxiety↗
DOE OSTI2025

Genetic variation at transcription factor binding sites largely explains phenotypic heritability in maize

Abstract Comprehensive maps of functional variation at transcription factor (TF) binding sites (cis-elements) are crucial for elucidating how genotype shapes phenotype. Here, we report the construction of a pan-cistrome of the maize leaf under well-watered and drought conditions. We quantified haplotype-specific TF footprints across a pan-genome of 25 maize hybrids and mapped over 200,000 variants, genetic, epigenetic, or both (termed binding quantitative trait loci (bQTL)), linked tocis-element occupancy. Three lines of evidence support the functional significance of bQTL: (1) coincidence with causative loci that regulate traits, includingvgt1,ZmTRE1and the MITE transposon nearZmNAC111under drought; (2) bQTL allelic bias is shared between inbred parents and matches chromatin immunoprecipitation sequencing results; and (3) partitioning genetic variation across genomic regions demonstrates that bQTL capture the majority of heritable trait variation across ~72% of 143 phenotypes. Our study provides an auspicious approach to make functionalcis-variation accessible at scale for genetic studies and targeted engineering of complex traits.

Genetics & Heredity↗
DOE OSTI2024

Genetic Basis of Non–Photochemical Quenching and Photosystem II Efficiency Responses to Chilling in the Biomass Crop Miscanthus

Miscanthus holds a promise as a biocrop due to its high yield, perenniality and ability to grow on infertile soils. However, the current commercial biomass production of Miscanthus is mostly limited to a single sterile triploid clone of M. × giganteus. Nevertheless, parental species of M. × giganteus, Miscanthus sacchariflorus and Miscanthus sinensis contain vast genetic diversity for crop improvement. With M. sacchariflorus having a natural geographic distribution in cold-temperate northeast China and eastern Russia, we hypothesised that it has substantial variation in physiological response to chilling. Using a semi-high-throughput method, we phenotyped 209 M. sacchariflorus genotypes belonging to six genetic groups for non-photochemical quenching (NPQ) and photosystem II efficiency (ΦPSII) kinetics under warm and chilling treatments in three growing seasons. In response to the chilling treatment, all genetic groups exhibited an increase in NPQ induction rate indicating faster activation of NPQ in light. Notably, under chilling, the Korea/NE China/Russia 2x and N China 2x groups stood out for the highest NPQ rate in light and the highest steady-state NPQ in light. This NPQ phenotype may contribute adaptation to chilling during bright, cold mornings of spring and early autumn in temperate climates, when faster NPQ would better protect from oxidative stress. Such enhanced adaptation could expand the growing season and thus productivity at a given location or expand the range of economically viable growing locations to higher latitudes and altitudes. A genome-wide association study identified 126 unique SNPs associated with NPQ and ΦPSII traits. Among the identified candidate genes were enzymes involved in the ascorbate recycle and shikimate pathway, gamma-aminobutyric acid and cation efflux transporters. Identifying natural variation and genes involved in NPQ and ΦPSII kinetics considerably enlarges the toolbox for breeding and/or engineering Miscanthus with optimised photosynthesis under warm and chilling conditions for sustainable feedstock production for bioenergy.

09 BIOMASS FUELS↗
DOE OSTI2025

Spatial Replication Is Important for Developing Landscape Genetic Inferences for a Wetland Salamander

Habitat fragmentation is a pressing threat to wildlife populations, and maintenance of gene flow between populations is an essential goal of conservation. Resistance surfaces have emerged as an important tool for modelling connectivity and developing management strategies to mitigate effects of habitat fragmentation. However, recent studies have noted inconsistencies in the factors most strongly associated with connectivity across different landscapes. Thus, replication of genetic-based resistance surface optimisation across landscapes may be necessary for making robust conclusions about the influence of environmental variables. Accordingly, replication represents a substantive challenge and opportunity in the field of landscape genetics. In this study, we conducted replicated landscape genetic analyses across five landscapes in Tennessee and Kentucky for a threatened wetland amphibian, the four-toed salamander (Hemidactylium scutatum). We tested multiple hypotheses of how different landscape features that could directly affect small, desiccation-intolerant amphibians (e.g., canopy cover) influenced gene flow and assessed the appropriate scale at which to model different features. We found some concordance in the landscape features that influenced gene flow (e.g., a common importance of forest cover and topography), but also some differences—potentially owing to the difference in variability of predictors across landscapes. We also found discordance in the scale of effect of different features across landscapes. In conclusion, our work emphasises that flat areas of moist forest not bisected by roads may be important for H. scutatum conservation, and our replicated design allows us to identify relationships that would have been missed if only using one study site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Chromosome-level genome assemblies and genetic maps reveal heterochiasmy and macrosynteny in endangered Atlantic Acropora

Abstract Background Over their evolutionary history, corals have adapted to sea level rise and increasing ocean temperatures, however, it is unclear how quickly they may respond to rapid change. Genome structure and genetic diversity contained within may highlight their adaptive potential. Results We present chromosome-scale genome assemblies and linkage maps of the critically endangered Atlantic acroporids,Acropora palmataandA. cervicornis. Both assemblies and linkage maps were resolved into 14 chromosomes with their gene content and colinearity. Repeats and chromosome arrangements were largely preserved between the species. The family Acroporidae and the genusAcroporaexhibited many phylogenetically significant gene family expansions. Macrosynteny decreased with phylogenetic distance. Nevertheless, scleractinians shared six of the 21 cnidarian ancestral linkage groups as well as numerous fission and fusion events compared to other distantly related cnidarians. Genetic linkage maps were constructed from oneA. palmatafamily and 16A. cervicornisfamilies using a genotyping array. The consensus maps span 1,013.42 cM and 927.36 cM forA. palmataandA. cervicornis, respectively. Both species exhibited high genome-wide recombination rates (3.04 to 3.53 cM/Mb) and pronounced sex-based differences, known as heterochiasmy, with 2 to 2.5X higher recombination rates estimated in the female maps. Conclusions Together, the chromosome-scale assemblies and genetic maps we present here are the first detailed look at the genomic landscapes of the critically endangered Atlantic acroporids. These data sets revealed that adaptive capacity of Atlantic acroporids is not limited by their recombination rates. The sister species maintain macrosynteny with few genes with high sequence divergence that may act as reproductive barriers between them. In the AtlanticAcropora, hybridization between the two sister species yields an F1 hybrid with limited fertility despite the high levels of macrosynteny and gene colinearity of their genomes. Together, these resources now enable genome-wide association studies and discovery of quantitative trait loci, two tools that can aid in the conservation of these species.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

BrainXcan identifies brain features associated with behavioral and psychiatric traits using large-scale genetic and imaging data

Advances in brain MRI have enabled many discoveries in neuroscience. Case-control comparisons of brain MRI features have highlighted potential causes of psychiatric and behavioral disorders. However, due to the cost and difficulty of collecting MRI data, most studies have small sample sizes, limiting their reliability. Furthermore, reverse causality complicates interpretation because many observed brain differences are the result rather than the cause of the disease. Here we propose a method (BrainXcan) that leverages the power of large-scale genomewide association studies (GWAS) and reference brain MRI data to discover new mechanisms of disease etiology and validate existing ones. BrainXcan tests the association with genetic predictors of brain MRI-derived features and complex traits to pinpoint relevant brain-wide and region-specific features. Requiring only genetic data, BrainXcan allows us to test a host of hypotheses on mental illness, across many MRI modalities, using public data resources. For example, our method shows that reduced axonal density across the brain is associated with schizophrenia risk, consistent with the disconnectivity hypothesis. We also find that the hippocampus volume is associated with schizophrenia risk, highlighting the potential of our approach. Taken together, our results show the promise of BrainXcan to provide insights into the biology of GWAS traits.

Association study↗
DOE OSTI2025

Nonenzymatic RNA copying with a potentially primordial genetic alphabet

Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U because of the stronger G:C vs. A:U base pair and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s 2 U) and inosine (I) form by deamination of 2-thiocytidine (s 2 C) and A, respectively. We used thermodynamic and crystallographic analyses to compare the I:s 2 C and A:s 2 U base pairs. We find that the I:s 2 C base pair is isomorphic and isoenergetic with the A:s 2 U base pair. The I:s 2 C base pair is weaker than a canonical G:C base pair, while the A:s 2 U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s 2 U, s 2 C, I, and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s 2 C and s 2 U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s 2 U, s 2 C, I, and A and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying.

Science & Technology - Other Topics↗
DOE OSTI2025

Unveiling shared genetic regulators of plant architectural and biomass yield traits in the Sorghum Association Panel

Abstract Sorghum is emerging as an ideal genetic model for designing high-biomass bioenergy crops. Biomass yield, a complex trait influenced by various plant architectural characteristics, is typically regulated by numerous genes. This study aimed to dissect the genetic regulators underlying 14 plant architectural traits and 10 biomass yield traits in the Sorghum Association Panel across two growing seasons. We identified 321 associated loci through genome-wide association studies (GWAS), involving 234 264 single nucleotide polymorphisms (SNPs). These loci include genes with known associations to biomass traits, such as maturity, dwarfing (Dw), and leafbladeless1, as well as several uncharacterized loci not previously linked to these traits. We also identified 22 pleiotropic loci associated with variation in multiple phenotypes. Three of these loci, located on chromosomes 3 (S03_15463061), 6 (S06_42790178; Dw2), and 9 (S09_57005346; Dw1), exerted significant and consistent effects on multiple traits across both growing seasons. Additionally, we identified three genomic hotspots on chromosomes 6, 7, and 9, each containing multiple SNPs associated with variation in plant architecture and biomass yield traits. Chromosome-wise correlation analyses revealed multiple blocks of positively associated SNPs located near or within the same genomic regions. Finally, genome-wide correlation-based network analysis showed that loci associated with flowering, plant height, leaf traits, plant density, and tiller number per plant were highly interconnected with other genetic loci influencing plant architectural and biomass yield traits. The pyramiding of favorable alleles related to these traits holds promise for enhancing the future development of bioenergy sorghum crops.

Singh, Anuradha (ORCID:0000000197149095)↗
DOE OSTI2025

Genetic Identification of Lamprey Genera and Anadromous Ecotypes in Watersheds of the Northeastern Pacific Ocean

ABSTRACT Nonparasitic, nonmigratory Western Brook Lamprey (WBL; Lampetra ayresii ), and parasitic, anadromous Western River Lamprey (WRL; L. ayresii ) are sympatric lampreys that likely represent different life history variations of a single species. Novel genetic tools are critical for differentiating WBL and WRL, whose larvae preclude morphological identification (ID) and will enable comprehensive assessment of imperiled native lampreys of the Northeastern Pacific (including WBL, WRL, and Pacific Lamprey, Entosphenus tridentatus ). We developed 47 candidate single nucleotide polymorphism (SNP) markers using whole genome resequencing of WBL ( N = 24) and WRL ( N = 15) from Ksi Ts'oohl Ts'ap Creek (Nass River, British Columbia, Canada) which are likely ecotypes distinguished by few divergent SNPs across multiple chromosomes. We used five novel candidate SNPs to perform genetic ID of WBL and WRL ecotypes in collections of mixed native lampreys from lower Columbia River tributaries ( N = 1474), Ksi Ts'oohl Ts'ap Creek ( N = 352), and ocean phase WRL from the Georgia Basin (Salish Sea, British Columbia, Canada; N = 91). Two previously published SNPs were used to ID genera, Entosphenus versus Lampetra . Morphological ID utilized photographs collected from a subset of genotyped lampreys, and high concordance was demonstrated between ID methods for genera (99%) and Lampetra ecotypes (> 98%). We characterized spatial and temporal composition of lamprey genera and ecotypes surveyed across NE Pacific tributaries under the expectation these compositions would be similar across nearby sites and across years at the same site. Proportions of lamprey genera were highly variable within regions and across years; however, Lampetra ecotypic proportions were spatially and temporally stable. WRL were rare in lower Columbia tributaries (~1% average rate among Lampetra ) and common further north (> 40% of Lampetra ). Genetic ID methods are powerful monitoring tools that create the novel ability to ascertain genera and ecotypes regardless of life stage, while increasing the efficiency of surveys by eliminating time‐intensive morphological data collection.

Silver, G. S. [Columbia River Inter‐Tribal Fish Co↗
DOE OSTI2024

Biomass yields, reproductive fertility, compositional analysis, and genetic diversity of newly developed triploid giant miscanthus hybrids

Abstract Miscanthus × giganteus (giant miscanthus), first found as a naturally occurring hybrid, has shown promise as a bioenergy/biomass crop throughout much of the temperate world. This allotriploid (2 n = 3 x = 57) hybrid resulted from a cross between tetraploid Miscanthus sacchariflorus (2 n = 4 x = 76) and diploid Miscanthus sinensis (2 n = 2 x = 38) and is particularly desirable due to its low fertility that minimizes reseeding and potential invasiveness. However, there is limited genetic diversity in commonly grown cultivars of triploid M. × giganteus and breeding and development efforts to improve and domesticate this crop have been minimal. Here, we report on newly developed M. × giganteus hybrids compared with the industry standard M. × giganteus '1993‐1780'. Dry biomass yields of new hybrids ranged from 19.5 to 32.4 Mg/ha/year for the fourth growing season, compared with 21.0 Mg/ha/year for M. × giganteus '1993‐1780'. Plant reproductive fertility remained low for all accessions with overall fertility [(seed set × seed germination)/100] ranging from 0.3% to 4.5% for new hybrids compared to 0.4% for M. × giganteus '1993‐1780'. Culm density and height varied among accessions and were positively correlated with increased biomass. Based on compositional analyses, theoretical ethanol yields ranged from 9, 740 to 16,278 L/ha/year for new hybrids compared to 10,406 L/ha/year for M. × giganteus '1993‐1780'. Relative feed value indices were low overall and ranged between 66.0 and 72.8 for new hybrids compared to M. × giganteus '1993‐1780' with 71.3. The genetic diversity of new hybrids, compared with existing cultivars, was characterized using whole genome sequences. Based on pair‐wise distances, cluster analysis clearly showed increased diversity of new hybrids compared with earlier selections. These results document new triploid hybrids of M. × giganteus with enhanced biomass and theoretical ethanol yields in combination with broader genetic diversity and lowreproductive fertility.

Touchell, Darren H.↗
DOE OSTI2026

Development of male-sterile lines of Setaria viridis to accelerate C 4 model plant genetics

Setaria viridis is a diploid C 4 grass in the Poaceae family, notable for its rapid life cycle of 6–8 weeks from sowing to seed—much shorter than the 4–5 months required by crops such as Zea mays and Sorghum bicolor . This fast growth makes S. viridis a valuable model for C 4 crop research. Genetic crosses are essential for studying gene function, but manual crossing is labor-intensive and time-consuming. Here, to address this, we developed a male-sterile line by targeting the S. viridis ortholog of Setaria italica NO POLLEN 1 ( SiNP1 ), which encodes a glucose–methanol–choline oxidoreductase required for pollen exine formation. Using Cas9 and TREX2 -mediated genome editing, we generated SiNP1 knockouts in both the S. viridis ME034V and A10.1 backgrounds that were fully male-sterile. Backcrossing T 0 male-sterile plants to ME034V wild-type followed by selfing yielded a stable BC 1 F 2 line homozygous for a 59 bp deletion in the S. viridis NO POLLEN 1 gene, easily genotyped by PCR and maintained by heterozygous siblings. Using this line, we developed a simple and efficient crossing protocol that eliminates the need for emasculation. This method enables a single person to perform up to 100 crosses per day—compared to 15 using traditional methods—and yields 20–32 F 1 hybrid seeds per panicle with 100% genetic purity. We also quantified pollen flow and outcrossing frequencies under greenhouse conditions to develop optimal bagging strategies and prevent unintended pollination. This resource accelerates genetic research in S. viridis , enhancing its utility as a premier C 4 model for mapping and functional genomics.

C4 research↗
DOE OSTI2024

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

ABSTRACT Leptospirosis, caused by pathogenic bacteria from the genusLeptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation ofLeptospirahas been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal toLeptospiracells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenicLeptospirafrom multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulateLeptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenicLeptospiraspp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system toLeptospiraenhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique inLeptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in theLeptospira borgpeterseniibackground, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms ofLeptospiraand the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Microbiology↗
DOE OSTI2024

Improving precision and accuracy of genetic mapping with genotyping-by-sequencing data in outcrossing species

This dataset contains all data and supplementary materials from "Improving precision and accuracy of genetic mapping with genotyping-by-sequencing data in outcrossing species". An Excel file a list of all QTLs and linkage group length (in cM) obtained with two different SNP-calling methods (Tassel-Uneak and Tassel-GBS), genetic map-construction method (linkage-only and reference order-corrected) and depth filters (12x, 20x, 30x and 40x) for genetic mapping of 18 biomass yield traits in a biparental Miscanthus sinensis population using RAD-Seq SNPs is provided as "Supplementary file 1". A Perl script with the code for filtering VCF and HapMap-formatted data files is provided as “Supplementary file 2”. Phenotype data used for QTL mapping is provided as “Supplementary File 3”. A Perl script with the code for the simulation study is provided as “Supplementary file 4”.

GenotypingSimulator↗
DOE OSTI2025

Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations

Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1, required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. Furthermore, this study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗