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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO 2 - ) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. Here, the results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH 3 , and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Pond Crash Forensics: Presumptive identification of pond crash agents by next generation sequencing in replicate raceway mass cultures of Nannochloropsis salina

Productivity of algal mass culture can be severely reduced by contaminating organisms. It is, therefore, important to identify contaminants, determine their effect on productivity and, ultimately, develop countermeasures against such contamination. In this paper, we utilized microbiome analysis by second-generation sequencing of small subunit rRNA genes to characterize the predator and pathogen burden of open raceway cultures of Nannochloropsis salina. Samples were analyzed from replicate raceways before and after crashes. In one culture cycle, we identified two algivorous species, the rotifer Brachionus and gastrotrich Chaetonotus, the presence of which may have contributed to the loss of algal biomass. In the second culture cycle, the raceways were treated with hypochlorite in an unsuccessful attempt to interdict the crash. Finally, our analyses were shown to be an effective strategy for the identification of the biological contaminants and the characterization of intervention strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

SU-E-T-45: Antibody Mean Residence Time in Blood and Its Correlation with Protein Molecular Weight

Purpose: Animal biodistribution data are required prior to introducing a new radiopharmaceutical into clinical trials. Protein engineering, using recombinant DNA techniques can produce a large number of related (cognate) antibodies to a given molecular target. Thus, it is important that these constructs be numerically related to one another via a single criterion. In the following, we use the mean residence time (MRT) in murine blood as this criterion. Methods: Five cognate anti-CEA (Carcinoembryonic Antigen) antibodies were compared with regard to their MRT in whole blood of CEA-positive tumor-bearing (LS174T) mice. MRT was defined by blood AUC (area under the curve) divided by the initial blood uptake value; all in units of percent injected dose per gram (%ID/g). Cognates included single chain scFv (25 kDa), diabody (50 kDa), minibody (80 kDa), F(ab')2 (120 kDa), and intact (155 kDa) forms of the murine cT84.66 antibody against CEA. All were labeled with radioactive iodine. Results: The agents, in the sequence listed, exhibited MRT values of 1.16 +/- 0.01 h, 0.99 h, 5.06 +/- 0.70 h, 6.61 +/- 0.36 h, and 59.3 +/- 2.4 h respectively. Because of the monotonic nature of the sequence, a linear correlation analysis was performed between molecular weight (MW) and MRT or ln(MRT) of the 5 proteins. Probability of random correlation was 0.10 for MRT and 0.01 for ln(MRT). Conclusion: MRT values of cognate anti-CEA antibodies were found to be a monotonically increasing sequence with respect to MW. Cognate MW values correlated best to ln(MRT) of the protein species. Thus MRT was proportional to an exponential function of molecular weight. The extended intact antibody circulation time presumably reflected its relatively maximal MW. Presence of an intact FC segment on this native antibody may also have influenced these results.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass)

Background: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass ( Panicum virgatum ), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass. Results: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession “Alamo”, upland accession “Dacotah”, and two upland/lowland F 1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors. Conclusion: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1975

Post-test navigation data analysis techniques for the shuttle ALT

Postflight test analysis data processing techniques for shuttle approach and landing tests (ALT) navigation data are defined. Postfight test processor requirements are described along with operational and design requirements, data input requirements, and software test requirements. The postflight test data processing is described based on the natural test sequence: quick-look analysis, postflight navigation processing, and error isolation processing. Emphasis is placed on the tradeoffs that must remain open and subject to analysis until final definition is achieved in the shuttle data processing system and the overall ALT plan. A development plan for the implementation of the ALT postflight test navigation data processing system is presented. Conclusions are presented.

Source record↗
NASA NTRS2008

Preparing Cassini Uplink Operations for Extended Mission

The Cassini-Huygens Mission to Saturn and Titan, a joint venture between the National Aeronautics and Space Administration, the European Space Agency, and the Italian Space Agency, is conducting a four-year, prime mission exploring the Saturnian system, including its atmosphere, rings, magnetosphere, moons and icy satellites. Launched in 1997, Cassini began its prime mission in 2004. Cassini is now preparing for a new era, a two-year extended mission to revisit many of the highlights and new discoveries made during the prime mission. Because of the light time delay from Earth to Saturn, and the time needed to coordinate the complicated science and engineering activities that take place on the spacecraft, commanding on Cassini is done in approximately 40-day intervals known as sequences. The Cassini Uplink Operations team is responsible for the final development and validation of the pointing profile and instrument and spacecraft commands that are contained in a sequence. During this final analysis prior to uplink to the spacecraft, thorough and exact evaluation is necessary to ensure there are no mistakes during commanding. In order to perform this evaluation, complete and refined processes and procedures are fundamental. The Uplink Operations team is also responsible for anomaly response during sequence execution, a process in which critical decisions often are made in real-time. Recent anomalies on other spacecraft missions have highlighted two major risks in the operations process: (1) personnel turnover and the retirement of critical knowledge and (2) aging, outdated operations procedures. If other missions are a good barometer, the Cassini extended mission will be presented with a high personnel turnover of the Cassini flight team, which could lead to a loss of expertise that has been essential to the success of the prime mission. In order to prepare the Cassini Uplink Operations Team for this possibility and to continue to develop and operate safe science and engineering sequences, a review and major update of the current documentation and operations procedures was needed. This paper will address the changes made to extended mission sequence generation processes primarily due to new restrictions in spacecraft operating capability and lessons learned from prime mission. In addition, it will address the state of the prime mission operations procedures, the philosophy changes and updates that were made to those procedures in response to process improvement, and the validation of those new procedures through the training of current and new personnel. And lastly, it will address the lessons learned throughout prime mission and how the Uplink Operations team chose to incorporate those lessons into the working documentation and team knowledge. This incorporation was necessary to facilitate the success of the extended mission with potentially all new personnel at some point prior to the end of the mission.

Maxwell, Jennifer L.↗
DOE OSTI2020

The relation between lignin sequence and its 3D structure

Background: Lignin, the second most abundant biopolymer on earth, plays a major structural role in plants, conferring mechanical strength and regulating water conduction. Understanding the three-dimensional structure of lignin is important for fundamental reasons as well as engineering plants towards lignin valorization. Lignin lacks a specific primary sequence, making its average chemical composition the focus of most recent studies. However, it remains unclear whether the 3D structure of lignin molecules depends on their sequence. Methods: In this work, we performed all-atom molecular dynamics simulation of three S/G-lignin molecules with the same average composition but different sequence. Results: A detailed statistical analysis of the radius of gyration and relative shape anisotropy reveals that the lignin sequence has no statistically significant effect on the global three-dimensional structure. We found however, that homopolymers of C-lignin with the same molecular weight have smaller radii of gyration than S/G-lignin. We attribute this to lower hydroxyl content of C-lignin, which makes it more compact and rigid. Conclusions: The 3D structure of lignin is influenced by the overall content of monomeric units and interunit linkages and not by its precise primary sequence. General Significance: Lignin is assumed to not have a well-defined primary structure. The results presented here demonstrate there are no significant differences in the global 3D structure of lignin molecules with the same average composition but different primary sequence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Fluorescent Amplification of Next Generation Sequencing Libraries Tools (FA-NGS tools) v1

This is a software utility for calculating pooling volumes and graphing quality control data of next generation sequencing libraries amplified with fluorescent intercalating agents and monitored by real-time quantitative PCR (qPCR). The tool is used to calculate pooling volumes of individual next generation sequencing libraries based on experimentally determined fluorescence values. To do this, fluorescent values are used as a proxy for relative concentration to determine the ratio of transfer volumes. The output is a "transfer file" that specifies the transfer volume and destination well of a given library. The tool can also be used to assess the quality of individual next generation sequencing libraries by melting curve analysis. The output are small-multiples graphs of the melting curves of individual sequencing libraries according to the specified plate layout.

Garber, Megan↗
NASA NTRS2016

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

yeast↗
DOE OSTI2017

Analysis of Plant Pan-Genomes and Transcriptomes with GET_HOMOLOGUES-EST, a Clustering Solution for Sequences of the Same Species

The pan-genome of a species is defined as the union of all the genes and noncoding sequences found in all its individuals. However, constructing a pan-genome for plants with large genomes is daunting both in sequencing cost and the scale of the required computational analysis. A more affordable alternative is to focus on the genic repertoire by using transcriptomic data. Here, the software GET_HOMOLOGUES-EST was benchmarked with genomic and RNA-seq data of 19 Arabidopsis thaliana ecotypes and then applied to the analysis of transcripts from 16 Hordeum vulgare genotypes. The goal was to sample their pan-genomes and classify sequences as core, if detected in all accessions, or accessory, when absent in some of them. The resulting sequence clusters were used to simulate pan-genome growth, and to compile Average Nucleotide Identity matrices that summarize intra-species variation. Although transcripts were found to under-estimate pan-genome size by at least 10%, we concluded that clusters of expressed sequences can recapitulate phylogeny and reproduce two properties observed in A. thaliana gene models: accessory loci show lower expression and higher non-synonymous substitution rates than core genes. Finally, accessory sequences were observed to preferentially encode transposon components in both species, plus disease resistance genes in cultivated barleys, and a variety of protein domains from other families that appear frequently associated with presence/absence variation in the literature. These results demonstrate that pan-genome analyses are useful to explore germplasm diversity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2016

Vertical Structure of Phyllosphere Fungal Communities in a Tropical Forest in Thailand Uncovered by High-Throughput Sequencing

Phyllosphere fungi harbor a tremendous species diversity and play important ecological roles. However, little is known about their distribution patterns within forest ecosystems. We examined how species diversity and community composition of phyllosphere fungi change along a vertical structure in a tropical forest in Thailand. Fungal communities in 144 leaf samples from 19 vertical layers (1.28–34.4 m above ground) of 73 plant individuals (27 species) were investigated by metabarcoding analysis using Ion Torrent sequencing. In total, 1,524 fungal operational taxonomic units (OTUs) were detected among 890,710 reads obtained from the 144 leaf samples. Taxonomically diverse fungi belonging to as many as 24 orders of Ascomycota and 21 orders of Basidiomycota were detected, most of which inhabited limited parts of the lowest layers closest to the forest floor. Species diversity of phyllosphere fungi was the highest in the lowest layers closest to the forest floor, decreased with increasing height, and lowest in the canopy; 742 and 55 fungal OTUs were detected at the lowest and highest layer, respectively. On the layers close to the forest floor, phyllosphere fungal communities were mainly composed of low frequency OTUs and largely differentiated among plant individuals. Conversely, in the canopy, fungal communities consisted of similar OTUs across plant individuals, and as many as 86.1%–92.7% of the OTUs found in the canopy (≥22 m above ground) were also distributed in the lower layers. Overall, our study showed the variability of phyllosphere fungal communities along the vertical gradient of plant vegetation and environmental conditions, suggesting the significance of biotic and abiotic variation for the species diversity of phyllosphere fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Transcriptome analysis in switchgrass discloses ecotype difference in photosynthetic efficiency

Background: Switchgrass, a warm-season perennial grass studied as a potential dedicated biofuel feedstock, is classified into two main taxa – lowland and upland ecotypes – that differ in morphology and habitat of adaptation. But there is limited information on their inherent molecular variations. Results: Transcriptome analysis by RNA-sequencing (RNA-Seq) was conducted for lowland and upland ecotypes to document their gene expression variations. Mapping of transcriptome to the reference genome (Panicum virgatum v1. 1) revealed that the lowland and upland ecotypes differ substantially in sets of genes transcribed as well as levels of expression. Differential gene expression analysis exhibited that transcripts related to photosynthesis efficiency and development and photosystem reaction center subunits were upregulated in lowlands compared to upland genotype. On the other hand, catalase isozymes, helix-loop-helix, late embryogenesis abundant group I, photosulfokinases, and S-adenosyl methionine synthase gene transcripts were upregulated in the upland compared to the lowlands. At ≥100x coverage and ≥5% minor allele frequency, a total of 25,894 and 16,979 single nucleotide polymorphism (SNP) markers were discovered for VS16 (upland ecotype) and K5 (lowland ecotype) against the reference genome. The allele combination of the SNPs revealed that the transition mutations are more prevalent than the transversion mutations. Conclusions: The gene ontology (GO) analysis of the transcriptome indicated lowland ecotype had significantly higher representation for cellular components associated with photosynthesis machinery controlling carbon fixation. In addition, using the transcriptome data, SNP markers were detected, which were distributed throughout the genome. The differentially expressed genes and SNP markers detected in this study would be useful resources for traits mapping and gene transfer across ecotypes in switchgrass breeding for increased biomass yield for biofuel conversion.

09 BIOMASS FUELS↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2014

Complete genome sequence of Planctomyces brasiliensis type strain (DSM 5305 T ), phylogenomic analysis and reclassification of Planctomycetes including the descriptions of Gimesia gen. nov., Planctopirus gen. nov. and Rubinisphaera gen. nov. and emended descriptions of the order Planctomycetales and the family Planctomycetaceae

Planctomyces brasiliensis Schlesner 1990 belongs to the order Planctomycetales , which differs from other bacterial taxa by several distinctive features such as internal cell compartmentalization, multiplication by forming buds directly from the spherical, ovoid or pear-shaped mother cell and a cell wall consisting of a proteinaceous layer rather than a peptidoglycan layer. The first strains of P. brasiliensis , including the type strain IFAM 1448 T, were isolated from a water sample of Lagoa Vermelha, a salt pit near Rio de Janeiro, Brasil. This is the second completed genome sequence of a type strain of the genus Planctomyces to be published and the sixth type strain genome sequence from the family Planctomycetaceae . The 6,006,602 bp long genome with its 4,811 protein-coding and 54 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project. We study phylogenomic analyses that indicate that the classification within the Planctomycetaceae is partially in conflict with its evolutionary history, as the positioning of Schlesneria renders the genus Planctomyces paraphyletic. A re-analysis of published fatty-acid measurements also does not support the current arrangement of the two genera. A quantitative comparison of phylogenetic and phenotypic aspects indicates that the three Planctomyces species with type strains available in public culture collections should be placed in separate genera. Thus the genera Gimesia, Planctopirus and Rubinisphaera are proposed to accommodate P. maris, P. limnophilus and P. brasiliensis , respectively. Pronounced differences between the reported G + C content of Gemmata obscuriglobus, Singulisphaera acidiphila and Zavarzinella formosa and G + C content calculated from their genome sequences call for emendation of their species descriptions. Lastly, in addition to other features, the range of G + C values reported for the genera within the Planctomycetaceae indicates that the descriptions of the family and the order should be emended.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Draft genome sequence of marine alphaproteobacterial strain HIMB11, the first cultivated representative of a unique lineage within the Roseobacter clade possessing an unusually small genome

Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Draft Genome Sequence of Serratia sp. Strain ATCC 39006, a Model Bacterium for Analysis of the Biosynthesis and Regulation of Prodigiosin, a Carbapenem, and Gas Vesicles

Serratia sp. strain ATCC 39006 is a Gram-negative bacterium and a member of the Enterobacteriaceae that produces various bioactive secondary metabolites, including the tripyrrole red pigment prodigiosin and the β-lactam antibiotic 1-carbapenen-2-em- 3-carboxylic acid (a carbapenem). This strain is the only member of the Enterobacteriaceae known to naturally produce gas vesicles, as flotation organelles. Here we present the genome sequence of this strain, which has served as a model for analysis of the biosynthesis and regulation of antibiotic production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Enhanced Resistance Pines for Improved Renewable Biofuel and Chemical Production (Technical Report)

We completed phenotyping constitutive and inducible oleoresin flow across two seasons, constitutive resin canal number and density and wood terpene content in our ADEPT2 and CCLONES populations. We completed genetic association between 19 oleoresin phenotypes and a total of 523,192 SNP markers from ADEPT2 and 13,883 SNP markers in CCLONES using four mixed linear models. A total of 293 significant SNPs (FDR = 0.20) were identified. We used the MENTOR tool to mine mechanistic connections from a multiplex network constructed from poplar multi-omic data to construct a conceptual model for a subset of these significant SNPs. Our model contains 6 transcriptional regulators in addition to 3 monoterpene synthases. To generate more lines of evidence for these significant SNPs, we completed a time course RNAseq experiment after inducing vascular zone cells to differentiate into new resin canals with a methyl jasmonate treatment, a single nuclei RNAseq that identified differentiating resin canal epithelial cells and are completing analysis for a QTL study in a hybrid pine population. The time course identified 4634 significantly down and 1890 significantly up regulated transcripts after treatment with methyl jasmonate, an inducer of new resin canal formation in the vascular cambial meristem. To analyze this large set of differentially regulated genes, we created a predictive expression network and analyzed it with random walk restart using 6 seed genes coding for transcription factors regulating xylem differentiation in poplar. Of the top ranked 200 transcripts, 119 transcripts were significant differentially expressed supporting these transcripts as potential candidates regulating resin canal formation. Analysis of single nuclei sequencing of shoot tips that contain differentiating resin canals, identified 10 clusters. One cluster was highly enriched in transcripts coding for 9 of the enzymes in the MEP pathway 3 prenyl synthetases, and 3 monoterpene synthases strongly suggesting that this cluster represents resin canal epithelial cells. We are mining the additional transcripts to create a trajectory analysis. In summary, we have identified > 10 novel genes that are strongly supported candidates for further analysis in breeding lines and for genetic engineering over- and under- expressing lines to increase wood terpene content to improve resistance to insect and fungal pathogens while simultaneously increasing terpene supplies for renewable chemicals and biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Effects of Herbarium Specimen Characteristics on Short-Read NGS Sequencing Success in Nearly 8000 Specimens: Old, Degraded Samples Have Lower DNA Yields but Consistent Sequencing Success

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies.

54 ENVIRONMENTAL SCIENCES↗