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All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

Congruence of Clusters Defined By Whole Genome Sequencing and MALDI-TOF for Bacteria Isolated From Cleanrooms

Introduction: Oligotrophic conditions can render cleanrooms inhospitable to microbes. Despite these constraints, fungi and bacteria are frequently isolated from surfaces in astromaterials cleanrooms at the Johnson Space Center. Bacillus species are of particular concern because endospores belonging to this genus are resilient and can affect astromaterials. Current monitoring programs rely on 16S rRNA sequencing and the VITEK2 Compact system. These methods have limited power to resolve Bacillus species. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to the industry gold standard, whole genome sequencing (WGS). Methods: Based on 16S rRNA classification, we selected 14 isolates for analysis with MALDI-TOF and WGS. Mass spectra were generated with MALDI-TOF MS and processed with custom scripts to identify clusters of closely related isolates and calculate a matrix of pairwise cosine similarity scores. Hybrid Illumina and Nanopore sequencing were used to generate draft genomes. Pairwise similarity scores were calculated from these genomes based on the average amino acid identity (AAI) predicted from single copy core genes. Congruence of clustering between these methods, was assessed by calculating adjusted Rand and Wallace coefficients. Results: Clusters of species generated from MALDI-TOF MS showed good agreement of phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other in terms of predicted amino acid sequences consistently showed cosine similarities of mass spectra > 0.65 and, of the 9 clusters identified with WGS, 8 were identical with MALDI-TOF. This corresponds to an adjusted Rand index of 0.95 and a 95% confidence interval of 0.80 – 1.00 for adjusted Wallace coefficients. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.84) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. Conclusion: This study shows that MALDI-TOF and WGS exhibit a similar ability to delineate Bacillus species isolated from cleanrooms and taxonomic units described by these two methods are consistent with one another. Since MALDI-TOF MS is low in cost and high in throughput, this approach appears to be an ideal option for routine microbial monitoring and identifying Bacillus species.

Farnaz Mazhari↗
NASA NTRSDate not supplied

Genomic and Phenotypic Associations to Predict Human Sensitivity to SpaceRadiation

High-linear energy transfer (LET) ionizing radiation is a major health hazard for astronauts who will be exposed to galactic cosmic rays during upcoming lunar and Mars missions. Predicting and mitigating this risk requires understanding the factors underlying individual radiation sensitivity. We started to address this challenge by identifying the genomic and phenotypic associations with sensitivity to low and high-LET ionizing radiation ex vivo in over 750 healthy human donors. We exposed primary human blood mononuclear cells to simulated galactic cosmic ray components: 350MeV/n 28Si, 350MeV/n 40Ar and 600MeV/n 56Fe particles, at 1.1 and 3 particles/100 sq.m fluences, as well as 0.1 Gy and 1 Gy doses of gamma rays, and analyzed the outcomes at 4 and 24 hours post-irradiation. We quantified DNA damage and repair responses based on 53BP1+ radiation-induced foci formation, together with oxidative stress and changes in secreted factors including immune cytokines and exosomes. We also analyzed phenotypic associations with spontaneous DNA repair foci at baseline prior to irradiation. We identified an increase in spontaneous DNA repair associated with age and latent viral infection, and observed that human spontaneous DNA repair foci at baseline can serve as a negative predictor of DNA repair and immunoregulatory cytokine production after irradiation. Furthermore, we observed a wide variability of subject- and LET-dependent radiation responses, with radiation-induced DNA repair foci increasing by dose and LET, and high-LET particle radiation resulting in more residual DNA damage compared to low-LET particles and gamma rays. We have developed multiple metrics to quantify human radio sensitivity across the spectrum of conditions. Here we present their dependence on radiation quality and phenotypic variables, including an age-dependent decrease in DNA repair after irradiation, and genomic associations with radio sensitivity based on low-coverage whole genome sequencing. We anticipate that our work will pave the way for understanding the spectrum of human radio sensitivity and identifying targets for countermeasure development to reduce DNA and cellular damage and radiation carcinogenesis during deep space exploration.

GWAS radiation human↗
NASA NTRSDate not supplied

Genomic and Phenotypic Predictors of Human Ex Vivo Sensitivity to Simulated Deep Space Radiation

High-linear energy transfer (LET) ionizing radiation is a major health hazard for astronauts who will be exposed to galactic cosmic rays during upcoming lunar and Mars missions. Predicting and mitigating this risk requires understanding the factors underlying individual radiation sensitivity. We started to address this challenge by identifying the genomic and phenotypic associations with sensitivity to low and high-LET ionizing radiation ex vivo in over 750 healthy human donors. We exposed primary human blood mononuclear cells to simulated galactic cosmic ray components: 350 MeV/n 28Si, 350 MeV/n 40Ar and 600 MeV/n 56Fe particles, at 1.1 and 3 particles/100 sq.m fluences, as well as 0.1 Gy and 1 Gy doses of gamma rays, and analyzed the outcomes at 4 and 24 hours post-irradiation. We quantified DNA damage and repair responses based on 53BP1+ radiation-induced foci formation, together with oxidative stress and changes in secreted factors including immune cytokines and exosomes. We also analyzed phenotypic associations with spontaneous DNA repair foci at baseline prior to irradiation. We identified an increase in spontaneous DNA repair associated with age and latent viral infection, and observed that human spontaneous DNA repair foci at baseline can serve as a negative predictor of DNA repair and immunoregulatory cytokine production after irradiation. Furthermore, we observed a wide variability of subject- and LET-dependent radiation responses, with radiation-induced DNA repair foci increasing by dose and LET, and high-LET particle radiation resulting in more residual DNA damage compared to low-LET particles and gamma rays. We have developed multiple metrics to quantify human radiosensitivity across the spectrum of conditions. Here we present their dependence on radiation quality and phenotypic variables, including an age-dependent decrease in DNA repair after irradiation, and genomic associations with radiosensitivity based on low-coverage whole genome sequencing. We anticipate that our work will pave the way for understanding the spectrum of human radiosensitivity and identifying targets for countermeasure development to reduce DNA and cellular damage and radiation carcinogenesis during deep space exploration.

GWAS↗
NASA NTRSDate not supplied

Genomic and phenotypic predictors of human ex vivo sensitivity to simulated deep space radiation

High-linear energy transfer (LET) ionizing radiation is a major health hazard for astronauts who will be exposed to galactic cosmic rays during upcoming lunar and Mars missions. Predicting and mitigating this risk requires understanding the factors underlying individual radiation sensitivity. We started to address this challenge by identifying the genomic and phenotypic associations with sensitivity to low and high-LET ionizing radiation ex vivo in over 750 healthy human donors. We exposed primary human blood mononuclear cells to simulated galactic cosmic ray components: 350MeV/n (28)Si, 350MeV/n (40)Ar and 600MeV/n (56)Fe particles, at 1.1 and 3 particles/100µm^(2) fluences, as well as 0.1 Gy and 1 Gy doses of gamma rays, and analyzed the outcomes at 4 and 24 hours post-irradiation. We quantified DNA damage and repair responses based on 53BP1^(+) radiation-induced foci formation, together with oxidative stress and changes in secreted factors including immune cytokines and exosomes. We also analyzed phenotypic associations with spontaneous DNA repair foci at baseline prior to irradiation. We identified an increase in spontaneous DNA repair associated with age and latent viral infection, and observed that human spontaneous DNA repair foci at baseline can serve as a negative predictor of DNA repair and immunoregulatory cytokine production after irradiation. Furthermore, we observed a wide variability of subject- and LET-dependent radiation responses, with radiation-induced DNA repair foci increasing by dose and LET, and high-LET particle radiation resulting in more residual DNA damage compared to low-LET particles and gamma rays. We have developed multiple metrics to quantify human radiosensitivity across the spectrum of conditions. Here we present their dependence on radiation quality and phenotypic variables, including an age-dependent decrease in DNA repair after irradiation, and genomic associations with radiosensitivity based on low-coverage whole genome sequencing. We anticipate that our work will pave the way for understanding the spectrum of human radiosensitivity and identifying targets for countermeasure development to reduce DNA and cellular damage and radiation carcinogenesis during deep space exploration.

GWAS↗
DOE OSTI2026

Ecological and genomic variation in ectomycorrhizal fungal exploration types

Ectomycorrhizal fungi (EMF) produce mycelia with variable extension and complexity, which can be classified according to soil ‘exploration types’ (ETs). ETs have received attention as one of the few mycorrhizal trait frameworks, but without an empirical classification of ET functional diversity and environmental preferences, understanding and interpreting EMF biogeographic patterns has been difficult. We conducted a synthesis combining: comparative EMF genomics to describe functional divergence in decomposition and nutrient cycling genes across ETs; and EMF trait distribution modeling across continental Europe, pairing soil and root EMF surveys to establish biogeographic ET niche profiles. We demonstrate a signature of ETs encoded in EMF genomes, which is independent from phylogeny and linked to biomass production strategies. EMF ET relative abundances were separated by soil, root, and dominant tree leaf type habitats and exhibited unique correlations with forest biotic (e.g. plant productivity and plant pathogen densities) and abiotic (e.g. nitrogen deposition and soil pH) conditions. These findings support a theory that EMF niche partitioning can be partially explained by extraradical mycelial traits, with underlying variation in ET biogeography likely arising from distinct decomposition and nutrient cycling potentials. We also identify important limitations to this trait framework and provide a guided outlook for future research.

biogeography↗
DOE OSTI2025

Characterisation and comparative analysis of mitochondrial genomes of false, yellow, black and blushing morels provide insights on their structure and evolution

Morchella species have considerable significance in terrestrial ecosystems, exhibiting a range of ecological lifestyles along the saprotrophism-to-symbiosis continuum. However, the mitochondrial genomes of these ascomycetous fungi have not been thoroughly studied, thereby impeding a comprehensive understanding of their genetic makeup and ecological role. In this study, we analysed the mitogenomes of 30 Morchellaceae species, including yellow, black, blushing and false morels. These mitogenomes are either circular or linear DNA molecules with lengths ranging from 217 to 565 kbp and GC content ranging from 38% to 48%. Fifteen core protein-coding genes, 28–37 tRNA genes and 3–8 rRNA genes were identified in these Morchellaceae mitogenomes. The gene order demonstrated a high level of conservation, with the cox1 gene consistently positioned adjacent to the rnS gene and cob gene flanked by apt genes. Some exceptions were observed, such as the rearrangement of atp6 and rps3 in Morchella importuna and the reversed order of atp6 and atp8 in certain morel mitogenomes. However, the arrangement of the tRNA genes remains conserved. We additionally investigated the distribution and phylogeny of homing endonuclease genes (HEGs) of the LAGLIDADG (LAGs) and GIY-YIG (GIYs) families. A total of 925 LAG and GIY sequences were detected, with individual species containing 19–48HEGs. These HEGs were primarily located in the cox1, cob, cox2 and nad5 introns and their presence and distribution displayed significant diversity amongst morel species. These elements significantly contribute to shaping their mitogenome diversity. Overall, this study provides novel insights into the phylogeny and evolution of the Morchellaceae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Application of functional genomics for domestication of novel non-model microbes

Abstract With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genomic reconstruction of Bacillus anthracis from complex environmental samples enables high-throughput identification and lineage assignment in Pakistan

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

A.Br.047 Vollum↗
DOE OSTI2026

Comparative physiological and genomic characterization of a novel Nitrobacter vulgaris strain from a nitrate-contaminated subsurface

Nitrite-oxidizing bacteria (NOB) represent a crucial node in the global nitrogen cycle. By catalyzing the second step of nitrification—the oxidation of nitrite to nitrate to generate energy for growth—NOB activity controls the fate of nitrite (NO 2 - ) in aerobic environments. Despite thriving in diverse environments, including soils, freshwater, marine ecosystems, subsurface habitats, and water treatment systems, organisms capable of nitrite oxidation are confined to Nitrobacter, Nitrospira, Nitrospina, Nitrotoga, and a few other specific lineages. The genus Nitrobacter, recognized for its facultative heterotrophic metabolism, is often associated with high-nitrogen environments. Here, we report the physiological characterization of a novel strain, Nitrobacter vulgaris strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface. Growth inhibition experiments revealed that strain MLSD-S22 and the N. vulgaris type strain Z exhibited similar sensitivities to nitrite and nitrate, with nitrite being the most inhibitory. Microrespirometry demonstrated that the two N. vulgaris strains and Nitrobacter winogradskyi Nb-255 possessed higher affinities for nitrite and oxygen than previously reported for Nitrobacter, suggesting potential to compete in low-substrate environments. Long-read DNA sequencing provided a complete genome for strain MLSD-S22, revealing two plasmids and an intact nitrous oxide (N 2 O) reduction operon—an unexpected feature for Nitrobacter. While N 2 O reduction activity was not observed under the tested conditions, this discovery raises questions about the contribution of Nitrobacter NOB to the N 2 O sink. These findings broaden the physiological and genomic diversity of Nitrobacter, offering new insights into their adaptation strategies and providing a framework for future evaluation of their potential roles in nitrogen loss.

Nitrobacter↗
DOE OSTI2024

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗
DOE OSTI2025

Metagenome-assembled genomes from topsoils collected during NEON campaign in East River, CO (06/14/2018-06/28/2018)

The Watershed Function Science Focus Area (WF SFA) at Lawrence Berkeley National Lab is working to build a mechanistic understanding of the distribution and dynamics of biogeochemical processes in mountainous watersheds and their response to perturbation. In June 2018, the NEON (National Ecological Observatory Network) Airborne Observatory Platform (AOP) performed a taskable airborne imaging campaign to collect visible to shortwave infrared (VSWIR) imaging spectroscopy and LiDAR data across 330 km2 in the Upper East River at Crested Butte, CO. We conducted a parallel ground sampling campaign to sample vegetation traits, as well as soil physical, chemical, and microbiological characteristics. We collected these samples from 438 sites across 12 locations spanning much of the elevation, topographic, and geologic variability across the study area. A subset of 250 samples were used for soil metagenomics which is presented here. In addition, at each site, vegetation samples were collected to measure species-specific leaf water content and leaf mass area, foliar elemental composition and foliar CN stable isotope ratios. Soil samples were collected to measure soil physical properties which include bulk density and soil texture analysis. A suite of soil chemical properties was measured from the samples collected at each site, including pH, organic matter, concentrations exchangeable cations, total elemental composition, and the concentrations of extractable N pools (e.g. total free amino acids, ammonium, nitrate, dissolved organic N, and total dissolved N). Additionally, we have measured soil microbial biomass CN stoichiometry. Here, we present 1982 metagenome-assembled genomes (MAGs) for the bacterial and archaeal community from topsoil collected from during NEON 2018 campaign. All metagenomes were sequenced at JGI (Joint Genome Institute) (GOLD Study ID: Gs0149986). Metagenomes were assembled using JGI Metagenome Workflow (10.1128/mSystems.00804-20). The dataset includes (1) zip files for 1982 MAG fasta files (neon_genomes1-5.tar.gz, split into 5 tarballs to keep tarballs under 0.5 GB), (2) neon_Gs0149986_samples_soilproperties_metagenomes.csv: the sample information together with the accession numbers for the underlying metagenomes and the associated soil physical and chemical measurements in NMDC (National Microbiome Data Collaborative) compliant format, (3) neon_Gs0149986.kml: location bounding box file for the sampled locations, (4) samples.csv: sample metadata file used to register Internationall Generic Sample Numbers (IGSNs), (5) flmd.csv: file level metadata file, and (6) dd.csv: data dictionary file. This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

2018 NEON and 2025 CHESS Campaigns↗
DOE OSTI2025

Genome-scale Design and Engineering of Non-model Yeast Organisms for Production of Biofuels and Bioproducts

The overall goal of this project was to develop genome-scale design and engineering tools for two non-model yeast organisms including Rhodotorula toruloides and Issatchenkia orientalis to produce high-levels of fatty acids-derived products and organic acids, respectively. The project was performed between 9/15/2017 and 9/14/2024 (the last two-years were no-cost extensions). The team consisted of Huimin Zhao (Lead PI) and Christopher Rao (Co-PI) from the University of Illinois at Urbana-Champaign (UIUC), Costas Maranas (Co-PI) from the Pennsylvania State University, Joshua Rabinowitz (Co-PI) and Martin Wuhr (Co-PI) from Princeton University, and Yasuo Yoshikuni (Co-PI) from the DOE Joint Genome Institute. The team has made great progress in both tool development and fundamental understanding of these two non-model yeasts. In total, there were 40 research publications (one of them is still under review) and one patent application as well as numerous oral presentations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Complete genome sequence of Sphingobium yanoikuyae strain CC4533

We have isolated a new strain of Sphingobium yanoikuyae , which belongs to the class Alphaproteobacteria, order Sphingomonadales, and family Sphingomonadaceae. This carotenoid-producing strain is capable of degrading xenobiotics and is tolerant to toxic levels of six heavy metals. We have designated the newly isolated strain of S. yanoikuyae as S. yanoikuyae strain CC4533 (hereafter called strain CC4533) because it was isolated from a contaminated Tris-Acetate-Phosphate (TAP) medium culture plate of a green micro-alga Chlamydomonas reinhardtii wild type strain CC4533. We sequenced the whole genome of strain CC4533 using the PacBio Sequel II Continuous Long Read technology and have submitted it to NCBI along with the SRA and PacBio methylation motif data. Additionally, we have submitted the PacBio methylome to REBASE, Ref#35996. We present the whole genome sequence of S. yanoikuyae strain CC4533 that offers insights into its coding and non-coding genes and its nearest taxonomic neighbors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genomes of eight cultured microbes from soil sites in Wellesley, MA

We present the genomes of eight cultured microbes isolated from surface soil in Wellesley, MA. The dataset is useful for exploring genomic diversity among freshwater taxa including Pedobacter, Bacillus, Paenibacillus, Streptomyces, and Flavobacterium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Genome_shuffling_enables_quantitative_trait_locus_mapping_in_Bacillus_subtilis

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion inBacillus subtilisto generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size (∼10 kb), and these associations were validated through targeted genetic swaps. ThisB. subtilisQTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria.

Bacillus subtilis↗
DOE OSTI2024

Longitudinal genome-wide association study reveals early QTL that predict biomass accumulation under cold stress in sorghum

Sorghum bicolor is a promising cellulosic feedstock crop for bioenergy due to its high biomass yields. However, early growth phases of sorghum are sensitive to cold stress, limiting its planting in temperate environments. Cold adaptability is crucial for cultivating bioenergy and grain sorghum at higher latitudes and elevations, or for extending the growing season. Identifying genes and alleles that enhance biomass accumulation under early cold stress can lead to improved sorghum varieties through breeding or genetic engineering. We conducted image-based phenotyping on 369 accessions from the sorghum Bioenergy Association Panel (BAP) in a controlled environment with early cold treatment. The BAP includes diverse accessions with dense genotyping and varied racial, geographical, and phenotypic backgrounds. Daily, non-destructive imaging allowed temporal analysis of growth-related traits and water use efficiency (WUE). A genome-wide association study (GWAS) was performed to identify genomic intervals and genes associated with cold stress response. The GWAS identified transient quantitative trait loci (QTL) strongly associated with growth-related traits, enabling an exploration of the genetic basis of cold stress response at different developmental stages. This analysis of daily growth traits, rather than endpoint traits, revealed early transient QTL predictive of final phenotypes. The study identified both known and novel candidate genes associated with growth-related traits and temporal responses to cold stress. The identified QTL and candidate genes contribute to understanding the genetic mechanisms underlying sorghum's response to cold stress. These findings can inform breeding and genetic engineering strategies to develop sorghum varieties with improved biomass yields and resilience to cold, facilitating earlier planting, extended growing seasons, and cultivation at higher latitudes and elevations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Optimizing resource allocation in Miscanthus breeding via sparse testing designs for genomic prediction

Phenotyping high-biomass perennial crops is laborious and the rate of genetic gain in conventional perennial crop breeding programs is typically low. So, it is especially important to identify methods that produce efficiency gains in the breeding process. Miscanthus is a C4 perennial grass with favorable characteristics for producing biomass as a feedstock for biofuels and diverse bio-based products. Increasing biomass yield will increase profitability and environmental benefits, so it is a key target for Miscanthus breeding. In addition, the identification of well-adapted genotypes across a wide range of environmental conditions requires the establishment of multi-environment trials (METs). Sparse testing is a genomic prediction-based strategy that reduces the phenotyping costs in METs by selecting a subset of genotypes to evaluate in a subset of environments and then predicts the performance of the unobserved genotype-environment combinations. A Miscanthus sacchariflorus (MSA) population comprising 336 genotypes observed across three environments was analyzed implementing sparse testing designs. Three prediction models considering main effects (environments, genotypes, genomic) and interaction effects (genotype-by-environment; G×E interaction) were implemented for forecasting dry biomass yield (YDY), total culm (TCM), average internode length (AIL), and culm node number (CNN). Multiple calibration sets based on different compositions and sizes were considered to evaluate performance in terms of the predictive ability (PA) and the mean square error (MSE) for a fixed testing set size. The training set size ranged from 52 to 112 to predict a fixed set of 224 unobserved genotypes across all three environments. The results showed that the model accounting for G×E interaction consistently presented the highest PA and the lowest MSE: for CNN (PA: ~0.77, MSE: ~0.5) and YDY (PA: ~0.70, MSE: ~1.3) while for TCM and AIL these ranged from ~0.28 to 0.41 and ~1.3 to 4.3, respectively. Overall, varying training sets and allocation strategies did not affect PA and MSE, with 52 non-overlapping and 0 overlapping genotypes per environment as the optimal cost-effective allocation framework. This suggests that implementing sparse testing designs could significantly reduce phenotyping costs by fivefold, without compromising PA in breeding programs for perennial crops such as Miscanthus.

Miscanthus sacchariflorus (MSA)↗