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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

$\mathrm{CROPSR}$: an automated platform for complex genome-wide $\mathrm{CRISPR}$ g$\mathrm{RNA}$ design and validation

CRISPR/Cas9 technology has become an important tool to generate targeted, highly specific genome mutations. The technology has great potential for crop improvement, as crop genomes are tailored to optimize specific traits over generations of breeding. Many crops have highly complex and polyploid genomes, particularly those used for bioenergy or bioproducts. The majority of tools currently available for designing and evaluating gRNAs for CRISPR experiments were developed based on mammalian genomes that do not share the characteristics or design criteria for crop genomes. We have developed an open source tool for genome-wide design and evaluation of gRNA sequences for CRISPR experiments, CROPSR. The genome-wide approach provides a significant decrease in the time required to design a CRISPR experiment, including validation through PCR, at the expense of an overhead compute time required once per genome, at the first run. To better cater to the needs of crop geneticists, restrictions imposed by other packages on design and evaluation of gRNA sequences were lifted. A new machine learning model was developed to provide scores while avoiding situations in which the currently available tools sometimes failed to provide guides for repetitive, A/T-rich genomic regions. We show that our gRNA scoring model provides a significant increase in prediction accuracy over existing tools, even in non-crop genomes. CROPSR provides the scientific community with new methods and a new workflow for performing CRISPR/Cas9 knockout experiments. CROPSR reduces the challenges of working in crops, and helps speed gRNA sequence design, evaluation and validation. We hope that the new software will accelerate discovery and reduce the number of failed experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Abstract Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

1-2↗
DOE OSTI2017

Single-molecule sequencing and Hi-C-based proximity-guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution

Background: Amaranth (Amaranthus hypochondriacus) was a food staple among the ancient civilizations of Central and South America that has recently received increased attention due to the high nutritional value of the seeds, with the potential to help alleviate malnutrition and food security concerns, particularly in arid and semiarid regions of the developing world. Here, we present a reference-quality assembly of the amaranth genome which will assist the agronomic development of the species. Results: Utilizing single-molecule, real-time sequencing (Pacific Biosciences) and chromatin interaction mapping (Hi-C) to close assembly gaps and scaffold contigs, respectively, we improved our previously reported Illumina-based assembly to produce a chromosome-scale assembly with a scaffold N50 of 24.4 Mb. The 16 largest scaffolds contain 98% of the assembly and likely represent the haploid chromosomes (n = 16). To demonstrate the accuracy and utility of this approach, we produced physical and genetic maps and identified candidate genes for the betalain pigmentation pathway. The chromosome-scale assembly facilitated a genome-wide syntenic comparison of amaranth with other Amaranthaceae species, revealing chromosome loss and fusion events in amaranth that explain the reduction from the ancestral haploid chromosome number (n = 18) for a tetraploid member of the Amaranthaceae. Conclusions: The assembly method reported here minimizes cost by relying primarily on short-read technology and is one of the first reported uses of in vivo Hi-C for assembly of a plant genome. Our analyses implicate chromosome loss and fusion as major evolutionary events in the 2n = 32 amaranths and clearly establish the homoeologous relationship among most of the subgenome chromosomes, which will facilitate future investigations of intragenomic changes that occurred post polyploidization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Selection signatures in four lignin genes from switchgrass populations divergently selected for in vitro dry matter digestibility

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four loci in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. As a result, this study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.

09 BIOMASS FUELS↗
DOE OSTI2018

Final Report for “The Genomic Basis of Heterosis in High-Yielding Triploid Hybrids of Willow ( Salix spp.) Bioenergy Crops”

Many studies have highlighted the complex, multigenic basis for heterosis (hybrid vigor) in inbred crops. Despite the lack a consensus model, it is vital that we turn our attention to understanding heterosis in undomesticated, outcrossing, heterozygous, and often polyploid species, such as willow ( Salix ). Shrub willow is a dedicated energy crop and is bred to be fast-growing and high-yielding on marginal land without competing with food crops. A trend in willow breeding is the consistent pattern of heterosis in triploid progeny produced from crosses between diploid and tetraploid species. Critical in understanding heterosis, the heritability of gene expression is dependent on allele-specific expression by local and remote factors in the genome. Here, we tested whether differentially expressed genes are responsible for heterosis in triploid crosses made between diploid S. purpurea , diploid S. viminalis , and tetraploid S. miyabeana parents. Three biological replicates of progeny and parent shoot tips were collected after 11 weeks in the greenhouse and individually sequenced via RNA-Seq (2×101). Our results highlight regulatory factors influencing differential expression and top modules of co-expressed genes correlated with heterosis for phenotypes collected in the greenhouse and in the field. We also show in diploid F 1 and F 2 S. purpurea that expression-level dominance and sex dimorphic expression is pervasive in shoot tips. Gene expression in triploids largely matches expected dosage values, but there are interesting categories of genes whose expression is divergent from expected dosage of alleles. Candidate genes have been identified whose differential expression be confirmed via allele-specific assays. Altogether, these data will be used to develop predictive models of heterosis and complement the growing genomic resources available for the improvement of shrub willow bioenergy crops.

09 BIOMASS FUELS↗
DOE OSTI2018

Expanding the breeder’s toolbox for perennial grasses

With the increase in world energy demand and the environmental impacts associated with continued fossil fuel use, there is a great need for the development of alternative and sustainable fuel sources. Switchgrass ( Panicum virgatum L. ), a perennial grass native to North America, is poised to become one of the first dedicated biomass crops deployed on a large scale in the United States in large part due to its inherent high yield, adaptability and perennial nature. Current ‘cultivars’ of switchgrass are essentially undomesticated wild selections from natural populations. Thus, there is considerable potential for improvement of this emerging energy crop. Traditional breeding approaches in switchgrass have been hampered by its self-incompatibility and sensitivity to inbreeding, therefore there is a significant need to develop new tools for improved switchgrass breeding. The proposed research uses a systems biology approach to accelerate switchgrass breeding, to develop its biotechnological improvement through the generation of stress-tolerant varieties, and to establish ways to minimize the potential impact of transgenes on ecosystems. In this proposal, we will develop new breeding tools for perennial grasses and apply these tools toward improving switchgrass. Specifically, we will employ two complementary approaches to overcome the difficulty of breeding a large, polyploid, self-incompatible plant. The first approach will accelerate conventional breeding by developing a method to rapidly create doubled haploid (homozygous) lines for breeding. This approach is based on altering CENH3, a centromere-specific histone, leading to the production of haploid offspring when the centromere mutant is crossed to wild type plants. Subsequent genome doubling results in fully homozygous individuals and overcomes one of the major limitations in breeding self-incompatibile crops. These homozygous lines would allow switchgrass breeders to apply a variety of breeding approaches that have been previously impossible. The second approach is to develop tools and reagents to accelerate the biotechnological improvement of switchgrass including: modules for improved stress tolerance, a test bed to evaluate transgenes in a perennial model grass and a robust gene containment system. In order to maintain a favorable energy balance and avoid competition with food crops, dedicated biomass crops must be grown with minimal inputs (fertilizer and water) on marginal lands that are unsuited to the traditional high input agriculture necessary to produce food. Thus, abiotic stress tolerance and efficient water and nutrient use are of paramount importance. Considerable basic research has been directed at these areas and this knowledge has been used to develop transgenic approaches to greatly improve water and nutrient use efficiency. By applying these approaches to switchgrass we will rapidly increase drought tolerance and nutrient use efficiency. This approach can rapidly introduce these traits in different cultivars allowing breeders to add these traits to their elite germplasm. Since biotechnologically improved switchgrass must ultimately be grown in open fields, they must be designed to minimize the potential impact of the transgenes on non-target ecosystems. The best way to do this is to minimize gene flow from the transgenic plants. Thus, we will develop technology to introduce sterility into the transgenic plants. We will then combine this technology with the stress resistance modules to create switchgrass lines with enhanced resistance to abiotic stresses.

09 BIOMASS FUELS↗
DOE OSTI2018

Expanding the Breeder’s Toolbox for Perennial Grasses

With the increase in world energy demand and the environmental impacts associated with continued fossil fuel use, there is a great need for the development of alternative and sustainable fuel sources. Switchgrass (Panicum virgatum L.), a perennial grass native to North America, is poised to become one of the first dedicated biomass crops deployed on a large scale in the United States in large part due to its inherent high yield, adaptability and perennial nature. Current ‘cultivars’ of switchgrass are essentially undomesticated wild selections from natural populations. Thus, there is considerable potential for improvement of this emerging energy crop. Traditional breeding approaches in switchgrass have been hampered by its self-incompatibility and sensitivity to inbreeding, therefore there is a significant need to develop new tools for improved switchgrass breeding. The proposed research uses a systems biology approach to accelerate switchgrass breeding, to develop its biotechnological improvement through the generation of stress-tolerant varieties, and to establish ways to minimize the potential impact of transgenes on ecosystems. In this proposal, we will develop new breeding tools for perennial grasses and apply these tools toward improving switchgrass. Specifically, we will employ two complementary approaches to overcome the difficulty of breeding a large, polyploid, self-incompatible plant. The first approach will accelerate conventional breeding by developing a method to rapidly create doubled haploid (homozygous) lines for breeding. This approach is based on altering CENH3, a centromere-specific histone, leading to the production of haploid offspring when the centromere mutant is crossed to wild type plants. Subsequent genome doubling results in fully homozygous individuals and overcomes one of the major limitations in breeding self-incompatibile crops. These homozygous lines would allow switchgrass breeders to apply a variety of breeding approaches that have been previously impossible. The second approach is to develop tools and reagents to accelerate the biotechnological improvement of switchgrass including: modules for improved stress tolerance, a test bed to evaluate transgenes in a perennial model grass and a robust gene containment system. In order to maintain a favorable energy balance and avoid competition with food crops, dedicated biomass crops must be grown with minimal inputs (fertilizer and water) on marginal lands that are unsuited to the traditional high input agriculture necessary to produce food. Thus, abiotic stress tolerance and efficient water and nutrient use are of paramount importance. Considerable basic research has been directed at these areas and this knowledge has been used to develop transgenic approaches to greatly improve water and nutrient use efficiency. By applying these approaches to switchgrass we will rapidly increase drought tolerance and nutrient use efficiency. This approach can rapidly introduce these traits in different cultivars allowing breeders to add these traits to their elite germplasm. Since biotechnologically improved switchgrass must ultimately be grown in open fields, they must be designed to minimize the potential impact of the transgenes on non-target ecosystems. The best way to do this is to minimize gene flow from the transgenic plants. Thus, we will develop technology to introduce sterility into the transgenic plants. We will then combine this technology with the stress resistance modules to create switchgrass lines with enhanced resistance to abiotic stresses.

09 BIOMASS FUELS↗
DOE OSTI2020

Unraveling the Genetics of Two Key Biomass Traits that Differentiate Upland and Lowland Tetraploid Switchgrass Ecotypes, Colonization by Mycorrhizal Fungi and Frost Tolerance

The overall objectives of the proposal were to dissect the genetics of the ability of switchgrass to interact with arbuscular mycorrhizal fungi (AMF) and to tolerate subzero temperatures, and to investigate the effect of AMF colonization on freezing tolerance and biomass production. In order to achieve this, we generated genetic maps in an F2 mapping population derived from a cross between the lowland genotype AP13 and the upland genotype VS16. In addition to providing a framework for identification of quantitative trait loci (QTL), the genetic maps were used to validate and improve the switchgrass genome assembly. We mapped phenotypic quantitative trait loci (QTL) for AMF colonization and cold tolerance in the F 2 population. We demonstrated that identification of QTL for AMF colonization was achieved only if AMF genus-specific colonization levels were used as phenotypes. No QTL were obtained when the overall level of AMF colonization was used as a trait. We conducted RNASeq on a subset of the F2 progeny following cold acclimation, and developed statistical methods to conduct expression QTL (eQTL) analyses in a polyploid organism. A preliminary analysis of the results identified eQTL for a range of genes that were differentially regulated in the lowland AP13 compared to the upland VS16 under cold acclimation. Once sequencing of the transcriptomes of a temporal replicate of the mapping population that was cold-acclimated using the same conditions has been completed, a final eQTL analysis will be conducted.

09 BIOMASS FUELS↗
DOE OSTI2023

A Systems Approach to Increasing Carbon Flux to Seed Oil for Biofuels and Bioproducts Production in Camelina sativa (Final Report)

To combat climate change and alleviate the dependency of the United States on fossil fuels, the transition to biofuel crops has long been proposed as a crucial part of the long-term solution. Camelina sativa has emerged as one of the leading commercially viable options for biofuel and bioproduct production for the U.S. Camelina has the advantages of low agronomic inputs and natural resistance to diverse biotic and abiotic stresses relative to other oilseed crops, and Camelina oil-based blends have been tested and approved as liquid transportation fuels. A major limitation in the widespread adoption of Camelina as a viable industrial oilseed crop is its modest oil yields. This project directly investigated possible paths towards increasing oil, by employing tissue-specific and whole plant systems approaches to identify major regulatory bottlenecks. We developed a new high-throughput method for the identification of multi-gene transformants in polyploid species like Camelina sativa and used this to determine that, contrary to what mathematical models suggested, introduction of the microbial Entner-Doudoroff (ED) pathway into Camelina sativa did not result in significant seed oil increases. We developed flux maps providing accurate and statistically robust models of central carbon metabolism in Camelina, including of cultured Camelina embryos. This knowledge was used in the capacitation of a number of junior researchers in gaining knowledge on approaches used for quantitative flux map analyses. We identified several novel Camelina transcription factor genes regulating fatty acid biosynthesis that showed variable effects on seed oil accumulation in transgenic plants. Among a number of community resources, we also developed a knowledge base web resource (CamRegBase) to integrate Camelina gene regulatory information. Together, research outcomes from this project have contributed to a much better understanding of the regulation and bottlenecks to engineer seed oil production in Camelina.

09 BIOMASS FUELS↗
DOE OSTI2026

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

Altpeter, Fredy (ORCID:0000000208944976)↗
DOE OSTI2021

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2 n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

(Saccharum hybrid complex)↗
DOE OSTI2016

How Clonal Is Clonal? Genome Plasticity across Multicellular Segments of a “Candidatus Marithrix sp.” Filament from Sulfidic, Briny Seafloor Sediments in the Gulf of Mexico

“Candidatus Marithrix” is a recently described lineage within the group of large sulfur bacteria (Beggiatoaceae, Gammaproteobacteria). This genus of bacteria comprises vacuolated, attached-living filaments that inhabit the sediment surface around vent and seep sites in the marine environment. A single filament is ca. 100 µm in diameter, several millimeters long, and consists of hundreds of clonal cells, which are considered highly polyploid. Based on these characteristics, “Candidatus Marithrix” was used as a model organism for the assessment of genomic plasticity along segments of a single filament using next generation sequencing to possibly identify hotspots of microevolution. Using six consecutive segments of a single filament sampled from a mud volcano in the Gulf of Mexico, we recovered ca. 90% of the “Candidatus Marithrix” genome in each segment. There was a high level of genome conservation along the filament with average nucleotide identities between 99.98 and 100%. Different approaches to assemble all reads into a complete consensus genome could not fill the gaps. Each of the six segment datasets encoded merely a few hundred unique nucleotides and 5 or less unique genes—the residual content was redundant in all datasets. Besides the overall high genomic identity, we identified a similar number of single nucleotide polymorphisms (SNPs) between the clonal segments, which are comparable to numbers reported for other clonal organisms. An increase of SNPs with greater distance of filament segments was not observed. The polyploidy of the cells was apparent when analyzing the heterogeneity of reads within a segment. Here, a strong increase in single nucleotide variants, or “intrasegmental sequence heterogeneity” (ISH) events, was observed. These sites may represent hotspots for genome plasticity, and possibly microevolution, since two thirds of these variants were not co-localized across the genome copies of the multicellular filament.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative feedstock analysis in Setaria viridis L. as a model for C 4 bioenergy grasses and panicoid crop species

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus × giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

09 BIOMASS FUELS↗
DOE OSTI2023

Downregulation of barley ferulate 5-hydroxylase dramatically alters straw lignin structure without impact on mechanical properties

Barley is a major cereal crop for temperate climates, and a diploid genetic model for polyploid wheat. Cereal straw biomass is an attractive source of feedstock for green technologies but lignin, a key determinant of feedstock recalcitrance, complicates bio-conversion processes. However, manipulating lignin content to improve the conversion process could negatively affect agronomic traits. An alternative approach is to manipulate lignin composition which influences the physical and chemical properties of straw. This study validates the function of a barley ferulate 5-hydroxylase gene and demonstrates that its downregulation using the RNA-interference approach substantially impacts lignin composition. We identified five barley genes having putative ferulate 5-hydroxylase activity. Downregulation of HvF5H1 substantially reduced the lignin syringyl/guaiacyl (S/G) ratio in straw while the lignin content, straw mechanical properties, plant growth habit, and grain characteristics all remained unaffected. Metabolic profiling revealed significant changes in the abundance of 173 features in the HvF5H1-RNAi lines. The drastic changes in the lignin polymer of transgenic lines highlight the plasticity of barley lignification processes and the associated potential for manipulating and improving lignocellulosic biomass as a feedstock for green technologies. On the other hand, our results highlight some differences between the lignin biosynthetic pathway in barley, a temperate climate grass, and the warm climate grass, rice, and underscore potential diversity in the lignin biosynthetic pathways in grasses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Overcoming genetic paucity of Camelina sativa : possibilities for interspecific hybridization conditioned by the genus evolution pathway

Camelina or false flax (Camelina sativa) is an emerging oilseed crop and a feedstock for biofuel production. This species is believed to originate from Western Asian and Eastern European regions, where the center of diversity of the Camelina genus is located. Cultivated Camelina species arose via a series of polyploidization events, serving as bottlenecks narrowing genetic diversity of the species. The genetic paucity of C. sativa is foreseen as the most crucial limitation for successful breeding and improvement of this crop. A potential solution to this challenge could be gene introgression from Camelina wild species or from resynthesized allohexaploid C. sativa. However, both approaches would require a complete comprehension of the evolutionary trajectories that led to the C. sativa origin. Although there are some studies discussing the origin and evolution of Camelina hexaploid species, final conclusions have not been made yet. Here, we propose the most complete integrated evolutionary model for the Camelina genus based on the most recently described findings, which enables efficient improvement of C. sativa via the interspecific hybridization with its wild relatives. We also discuss issues of interspecific and intergeneric hybridization, aimed on improving C. sativa and overcoming the genetic paucity of this crop. The proposed comprehensive evolutionary model of Camelina species indicates that a newly described species Camelina neglecta has a key role in origin of tetra- and hexaploids, all of which have two C. neglecta-based subgenomes. Understanding of species evolution within the Camelina genus provides insights into further research on C. sativa improvements via gene introgression from wild species, and a potential resynthesis of this emerging oilseed crop.

09 BIOMASS FUELS↗
DOE OSTI2025

Gene editing to enhance biotic stress tolerance in sugarcane

Sugarcane (Saccharum spp.) contributes approximately 80% of global sugar production and 40% of biofuel while serving as a promising feedstock for bioproducts (Brant et al., 2025). However, productivity faces mounting challenges from biotic threats including fungal, viral and bacterial diseases (Rott, 2018). Traditional breeding approaches are severely constrained by sugarcane’s complex polyploid genome containing 10–12 copies of "hom(oe)ologous genes within an approximately 10 Gb genome (Healey et al., 2024), extending breeding cycles to 12–15 years. Genome editing has revolutionized crop improvement by enabling targeted modifications without necessarily introducing foreign DNA, potentially circumventing regulatory hurdles while accelerating variety development (Li et al., 2022a). For sugarcane, these technologies offer unprecedented opportunities to enhance biotic stress resilience while improving yield and quality. This article examines current progress and prospects for developing biotic stress-tolerant sugarcane through gene editing, emphasizing technical advances, promising gene targets, and strategic approaches for trait stacking.

CRISPR-Cas systems↗
DOE OSTI2016

Development of Microsatellite Markers for Buffalograss ( Buchloë dactyloides ; Poaceae), a Drought-Tolerant Turfgrass Alternative

Buchloë dactyloides (Nutt.) Engelm. (buffalograss; Poaceae) is a low-growing, perennial C4 grass that is a dominant component of shortgrass prairies of the North American Great Plains (Shearman et al., 2004). Beyond this significant ecosystem role, buffalograss has been widely adopted as a drought-tolerant turfgrass alternative, particularly notable as a native-species option in North America. Like many dominant Great Plains grasses, B. dactyloides comprises an autopolypoid series, including diploids (2n = 20), tetraploids, pentaploids, and hexaploids (Johnson et al., 2001). Preserving the full range of buffalograss phenotypic and genotypic diversity and utilizing this diversity for crop improvement will require an understanding of the distribution of genetic variation among cytotypes and across its large geographic range. Beyond numerous methodological advantages (Guichoux et al., 2011), microsatellites, or simple sequence repeat (SSR) markers,are an attractive genetic tool for studies of wide-ranging polyploid series given their codominant nature and applicability to museum-derived DNAs. Because SSR data are routinely obtainable from DNA extracted from museum tissue (Wandeler et al., 2007), these samples can be used to quickly and economically obtain comparative genotypic data from all portions of a large geographic range. Currently no buffalograss-specific SSR loci are available, as previous studies have relied on a mixture of dominant and codominant loci that were designed for other taxa (Budak et al., 2004). In this study, a set of SSR loci are designed from B. dactyloides genomic sequence data. The variability of these loci are then evaluated in six populations from numerous portions of the buffalograss range.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Data from: Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

Switchgrass (Panicum virgatum L.) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuels feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype composed of tetraploid and octoploid accessions, and a southern lowland ecotype composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single nucleotide polymorphisms (SNPs), of which 1,590,653 high confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype restricted non-synonymous SNPs that are predicted to impact protein function in genes that encode CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17,228 up-copy number variants (CNVs), 112,630 down-CNVs, and 14,430 presence/absence variants (PAV) impacting a total of 9,979 genes, including two upland-specific CNV-clusters. In total, 45,719 genes were impacted by a SNP, CNV, or a PAV across the panel providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.

09 BIOMASS FUELS↗