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At least 307 records · Page 17

Quantitative Trait Locus Mapping for Flowering Time in a Lowland × Upland Switchgrass Pseudo-F Population

Flowering is an important developmental event in switchgrass ( Panicum virgatum ), as the time to complete the life cycle affects overall biomass accumulation. The objective of this study was to generate a linkage map using single nucleotide polymorphism (SNP) markers to identify quantitative trait loci (QTL) associated with flowering time. A pseudo-F population was created by crossing two siblings derived from an initial cross between the lowland population Ellsworth and the upland cultivar Summer. Heading and anthesis dates were collected for 2 yr at two locations: DeKalb, IL and Lafayette, IN. Nine QTL for flowering time were detected, two of which were heading-associated, four anthesis-associated, and three associated with both heading and anthesis. One QTL on linkage group (LG) 2a was detected for heading and anthesis in each location and year when environments were analyzed separately, and in a combined analysis across both locations and years. The effect on heading and anthesis of the QTL on LG 2a ranged from 4 to 13 and 5 to 9 d, respectively, depending on environment. Our findings validate QTL for switchgrass flowering time from previous research and identified additional QTL. Based on the switchgrass reference genome version 1.1, flowering time gene homologs reside near the LG 2a QTL and include PSEUDO RESPONSE REGULATOR 5, SUPPRESSOR OF FRIGIDA 4, and APETALA 1, respectively involved in the circadian clock, vernalization, and floral meristem identity. Markers linked to the QTL can be used to improve the efficiency of breeding switchgrass for delayed flowering to increase biomass yield.

09 BIOMASS FUELS↗

Genome-Wide Association Mapping of Anthracnose ( Colletotrichum sublineolum ) Resistance in the U.S. Sorghum Association Panel

The productivity and profitability of sorghum [Sorghum bicolor (L.) Moench] is reduced by susceptibility to fungal diseases, such as anthracnose (Colletotrichum sublineolum P. Henn.). A limited number of resistant accessions are present in the temperate-adapted germplasm; other exotic sources of resistance are not currently available for breeding programs. Among 335 accessions available to breeders from a previously genotyped sorghum association panel (SAP), we found that 75 were resistant to anthracnose. A phylogenetic analysis of these accessions showed high genetic diversity and multiple resistance sources. Genome-wide association scans (GWAS) were conducted using 268,289 single-nucleotide polymorphisms to identify loci associated with anthracnose resistance. Using logistic regressions for binary measures of resistance responses, we identified three loci within a region on chromosome 5 that have been previously associated with three sources of anthracnose resistance. A GWAS limited to Caudatum germplasm identified an association with a region on chromosome 1 and with the same previous region on chromosome 5. Candidate genes within these loci were related to R-gene families, signaling cascades, and transcriptional reprogramming, suggesting that the resistance response is controlled by multiple defense mechanisms. The strategic integration of exotic resistant germplasm into the SAP is needed to identify additional rare resistance alleles via GWAS.

59 BASIC BIOLOGICAL SCIENCES↗

Multiple Maize Reference Genomes Impact the Identification of Variants by Genome-Wide Association Study in a Diverse Inbred Panel

Use of a single reference genome for genome-wide association studies (GWAS) limits the gene space represented to that of a single accession. This limitation can complicate identification and characterization of genes located within presence–absence variations (PAVs). In this study, we present the draft de novo genome assembly of ‘PHJ89’, an ‘Oh43’-type inbred line of maize (Zea mays L.). From three separate reference genome assemblies (‘B73’, ‘PH207’, and PHJ89) that represent the predominant germplasm groups of maize, we generated three separate whole-seedling gene expression profiles and single nucleotide polymorphism (SNP) matrices from a panel of 942 diverse inbred lines. We identified 34,447 (B73), 39,672 (PH207), and 37,436 (PHJ89) transcripts that are not present in the respective reference genome assemblies. Genome-wide association studies were conducted in the 942 inbred panel with both the SNP and expression data values to map Sugarcane mosaic virus (SCMV) resistance. Highlighting the impact of alternative reference genomes in gene discovery, the GWAS results for SCMV resistance with expression values as a surrogate measure of PAV resulted in robust detection of the physical location of a known resistance gene when the B73 reference that contains the gene was used, but not the PH207 reference. This study provides the valuable resource of the Oh43-type PHJ89 genome assembly as well as SNP and expression data for 942 individuals generated from three different reference genomes.

59 BASIC BIOLOGICAL SCIENCES↗

Genetic engineering and improvement of a Zymomonas mobilis for arabinose utilization and its performance on pretreated corn stover hydrolyzate

In this paper, a glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizing arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. Finally, we speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.

09 BIOMASS FUELS↗

Data from: Multiple maize reference genomes impact the identification of variants by GWAS in a diverse inbred panel

Use of a single reference genome for genome-wide association studies (GWAS) limits the gene space represented to that of a single accession. This limitation can complicate identification and characterization of genes located within presence/absence variations (PAVs). In this study, we present the draft de novo genome assembly of PHJ89, an Oh43-type inbred line. Using three separate reference genome assemblies (B73, PH207, and PHJ89) that represent the predominant germplasm groups of maize, we generated three separate whole-seedling gene expression profile and single nucleotide polymorphism (SNP) matrices from a panel of 942 diverse inbred lines. We identified 34,447 (B73), 39,672 (PH207), and 37,436 (PHJ89) transcripts that are not present in the respective reference genome assembly. GWAS was conducted in the 942 inbred panel using both the SNP and expression data values to map sugarcane mosaic virus (SCMV) resistance. Highlighting the impact of alternative reference genomes in gene discovery, GWAS results for SCMV resistance using expression values as a surrogate measure of PAV resulted in robust detection of the physical location of a known resistance gene when using the B73 reference that contains the gene, but not when using the PH207 reference. This study provides the valuable resource of the Oh43-type PHJ89 genome assembly as well as SNP and expression data for 942 individuals generated using three different reference genomes.

59 BASIC BIOLOGICAL SCIENCES↗

Data from: Extensive genetic diversity is present within North American switchgrass germplasm

Switchgrass (Panicum virgatum L.) is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice (Oryza sativa L.), maize (Zea mays L.), soybean [Glycine max (L.) Merr.], and Medicago truncatula Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass.

09 BIOMASS FUELS↗

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↗

Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18

Genome-wide association studies for non-syndromic orofacial clefting (OFC) have identified single nucleotide polymorphisms (SNPs) at loci where the presumed risk-relevant gene is expressed in oral periderm. The functional subsets of such SNPs are difficult to predict because the sequence underpinnings of periderm enhancers are unknown. We applied ATAC-seq to models of human palate periderm, including zebrafish periderm, mouse embryonic palate epithelia, and a human oral epithelium cell line, and to complementary mesenchymal cell types. We identified sets of enhancers specific to the epithelial cells and trained gapped-kmer support-vector-machine classifiers on these sets. We used the classifiers to predict the effects of 14 OFC-associated SNPs at 12q13 near KRT18. All the classifiers picked the same SNP as having the strongest effect, but the significance was highest with the classifier trained on zebrafish periderm. Reporter and deletion analyses support this SNP as lying within a periderm enhancer regulating KRT18/KRT8 expression.

59 BASIC BIOLOGICAL SCIENCES↗

Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths ( Bradypus variegatus ), the first from a non-human host

Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.

59 BASIC BIOLOGICAL SCIENCES↗

Microelectronic DNA assay for the detection of BRCA1 gene mutations

Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

Validation Studies↗

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility

Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the Martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07?×?10(7) spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8?h at ~31?km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240?min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480?min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1?nm UVC light source (0.5 W/m(2)), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions.

Planetary protection↗

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility

Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth’s stratosphere is severely cold, dry, irradiated and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the Martian surface. We launched E-MIST, a high altitude NASA balloon payload on 10 Oct 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 x107 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8 hours at ~32 km above sea level exposing bacterial spores to the stratosphere. We found that within 120 and 240 min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480 min, < 0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first Sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of ultraviolet (UV) light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and also conducted ground tests with a 271.1 nm UVC light source (0.5 W/m2), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and micro-configuration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a re-sequencing analysis and identified 3 single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth’s upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions.

Mars↗

Distribution and Relative Abundance of Scalloped (Sphyrna lewini) and Carolina (S. gilberti) Hammerheads in the Western Atlantic Ocean

The scalloped hammerhead (Sphyrna lewini) and its cryptic congener, Carolina hammerhead (S. gilberti), are sympatrically distributed in the western North Atlantic Ocean. Because the species are indistinguishable based on external morphology, little research focused on Carolina hammerheads exists. In this study, the distribution of Carolina hammerheads in waters of the United States off the east coast (U.S. Atlantic) and Gulf of Mexico (Gulf) was examined and their abundance relative to scalloped hammerheads assessed by genetically identifying 1,232 individuals using diagnostic single nucleotide polymorphisms. Both species were found in the U.S. Atlantic, where 27% of individuals were Carolina hammerheads, but only scalloped hammerheads were identified in the Gulf. In Bulls Bay, SC, a well-known hammerhead nursery, assessment of relative abundance from May to September showed scalloped hammerheads were more abundant May-June and Carolina hammerheads more abundant July-September. Results of this study suggest Carolina hammerheads have a spatially limited distribution in the western North Atlantic and highlight the importance of Bulls Bay as a nursery for the species. In addition, the results suggest Carolina hammerheads may comprise a non-trivial proportion of what is considered the U.S. Atlantic scalloped hammerhead stock and should be considered in future decisions regarding management of the hammerhead complex.

Sympatric↗

An Open-Science Approach to Address Individual Response to Simulated GCR In Genetically Diverse Populations of Mice and Humans

This project addresses the challenge of understanding and predicting individual radiation sensitivity by integrating genetics, demographics and biomarker characteristics across species (mice and humans). We hypothesize that ex vivo DNA repair response to GCR components is a central determinant of cancer risk from space radiation and can serve as a biomarker of radiation risk in combination with genetics. Automated image quantification of 53BP1+ radiation-induced foci (RIF) during the first 4-48 h post-irradiation was performed as a function of dose and LET in non-immortalized primary skin fibroblasts derived from 76 mice across 15 strains (5 inbred reference strains and 10 collaborative-cross strains) exposed to X rays (0.1, 1 and 4 Gy), 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100sq. μm), as well as in peripheral blood mononuclear cells (PBMCs) from 768 healthy donors (matched ethnicity, 50/50 male/female, 18-70 years old) exposed to gamma rays (0.1 and 1 Gy), 350 MeV/n 28Si, 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100sq. μm). A genome-wide association study (GWAS) was performed on the mouse strains between DNA damage responses to space radiation and single nucleotide polymorphisms (SNPs). We found SNPs, which were significantly associated to the RIF phenotype, mapped to genes and pathways that are functionally linked to health hazards for deep space exploration (e.g. carcinogenesis, nervous system damage and immune dysfunction). Some of these SNPs were located within protein coding regions, potentially interfering with protein functions and providing promising genetic targets for countermeasures. We also found correlations between both spontaneous and radiation-induced DNA damage and SNPs mapped to pathways associated with cellular metabolism. GWAS is undergoing for the human data. All data have been made available via the NASA Space Biology Open-Science database (genelab.nasa.gov) and we will discuss how various genomic and transcriptomic datasets can be accessed for modeling and integrated using machine learning methods for discovering new radiation biology.

Sylvain V Costes↗

One-Carbon Metabolism and SANS 2022 Update

Spaceflight Associated Neuro-ocular Syndrome, or SANS, affects a subset of astronauts (1, 2), and biochemical evidence has documented differences in those astronauts (3). Specifically, they had higher circulating concentrations of metabolites of the one-carbon metabolic pathway (1C), including homocysteine, and these concentrations were higher before flight (3). After ruling out many potential confounding factors in these otherwise healthy individuals (e.g., sex, kidney function, vitamin status, coffee consumption), a study of genetics was warranted. In an initial pilot effort, we documented a genetic predisposition to develop ophthalmic changes after long-duration space flight (4). That is, from a limited study of 5 single-nucleotide polymorphisms (SNPs), we found that the G allele for the MTRR A66G SNP was associated with a greater risk of choroidal folds and cotton-wool spots after flight, and the C allele for SHMT1 C1420T was protective against optic disc edema (4). These data provide a potential pathway for understanding why some individuals develop SANS, while others do not. The initial pilot study of 5 SNPs yielded striking findings, but the 1C pathway is far more complex. An effort was undertaken to examine more than 500 1C SNPs to see if a broader examination could help illuminate this association. That work is ongoing. The astronaut findings led us to advocate for the inclusion of 1C pathway genetic and biochemistry testing on other SANS-related projects, noting that genetics might help identify responders, non-responders, or outliers. The first such effort yielded evidence of an association of specific forms of the MTRR and SHMT-1 SNPs and vitamin B12 status with end-tidal CO2 after acute carbon dioxide exposure (5). The second such effort led to the identification that individuals exposed to strict head-down tilt and CO2 for 30-d who developed optic disc edema also had risk alleles for the two SNPs described above (6). Additionally, we identified a clinical population with many characteristics either attributed or purported to be involved in the ocular changes seen in affected astronauts: women with polycystic ovary syndrome (PCOS). PCOS is a condition of androgen excess and anovulatory menstrual cycles. The shared characteristics and clinical findings between SANS and PCOS generally include higher circulating homocysteine concentrations, increased retinal nerve fiber layer thickness, increased androgen concentrations (or responses), and altered carbohydrate metabolism. To our knowledge, no study has examined whether women with PCOS have asymptomatic ophthalmic anomalies observed in astronauts with SANS. While researchers have evaluated the one-carbon metabolism pathway polymorphisms of PCOS patients, and initial studies show an association with certain one-carbon polymorphisms, none have looked at the set of SNPs identified in our studies that are associated with ophthalmic changes in astronauts. Accordingly, we designed a study to evaluate the association of one-carbon pathway SNPs and ophthalmic findings in patients with PCOS and/or IIH compared to controls. Subjects provided blood samples for vitamin and one carbon biochemistry analyses, an extensive analysis of >500 SNPs associated with one carbon metabolism and had eye examinations and ocular imaging. Data analysis are underway. The data collected to date have shown associations between one carbon pathway biochemistry and genetics and incidence of SANS. The mechanisms for SANS has yet to be identified, although many hypotheses exist. Based on our data, we have developed (8, 9) and expanded (6) a multi-hit hypothesis for how these seemingly disparate findings could be linked. While intriguing, the hypothesis represents the starting point for further research. We aim to clarify the relationship between B-vitamin status and genetics with regard to the risk of SANS. Ultimately, understanding the mechanism(s) behind this will provide a means to predict, prevent, or treat these ophthalmologic pathologies in astronauts, and terrestrial populations.

S M Smith↗

Exploring Endothelial Function Risk Factors and Optic Disc Edema Changes During Strict 6º Head-Down Tilt Bed Rest

Approximately 20% of astronauts on International Space Station missions experience ophthalmic pathologies including optic disc edema, part of what is characterized as Spaceflight Associated Neuro-ocular Syndrome (SANS). While the cause of SANS is unknown, there are likely multiple contributing factors, including genetics, that may affect the response to spaceflight in affected individuals. B-vitamin status and the presence of specific one-carbon pathway single nucleotide polymorphism (SNP) alleles predicted the incidence of SANS pathologies in astronauts and in bed rest subjects. There are several hypotheses for how genetic variants could lead to SANS, specifically related to endothelial function. The biochemical pathway to which these genes are associated is intimately involved in maintaining endothelial function via nitric oxide synthase (eNOS) coupling and nitric oxide (NO) production. Furthermore, eNOS uncoupling can impair the functional status of the endothelial glycocalyx, which lines the entirety of the vascular lumen and affects endothelial function. Vascular distension, stasis and altered shear forces, which are associated with headward fluid shifts, may also contribute to glycocalyx dysfunction and shedding, resulting in endothelial dysfunction and increased vascular permeability and tissue edema, potentially contributing to optic nerve and optic disc edema in affected individuals. Given the findings relating genetics and the risk of optic disc edema in astronauts during flight and subjects during bed rest, further investigation is warranted. In this study, we will assess the same genetic variants in another bed rest study: AGBRESA. Some of the AGBRESA subjects developed optic disc edema, but their genetics have not been studied. Furthermore, we will test available urine samples from prior bed rest studies (VaPER and AGBRESA) for markers of glycocalyx degradation to expand our understanding of factors contributing to SANS.

S R Zwart↗

B Complex 5-Methyltetrahydrofolate, Riboflavin, Pyridoxine, and Methylcobalamin Supplementation as a Non-Mechanical Countermeasure to Mitigate Optic Disc Edema Changes During Strict 6º Head-Down Tilt Bed Rest

A subset of astronauts on International Space Station missions have experienced optic disc edema, part of what is characterized as Spaceflight Associated Neuro-ocular Syndrome (SANS). While the precise cause of SANS is unknown, it is likely that there are multiple contributing factors, including genetic and environmental factors. Our recent work has shown that crewmembers with SANS have higher concentrations of metabolic biomarkers of impairments in the one-carbon metabolic pathway compared to unaffected astronauts - before, during, and after f light (1). B-vitamin status and the presence of one-carbon pathway single nucleotide polymorphism (SNP) variants predicted the incidence of SANS pathologies, including optic disc edema (2). Specifically, the G allele of methionine synthase reductase (MTRR) A66G and the C allele of serine hydroxymethyltransferase-1 (SHMT1) C1420T were associated with increased incidence of SANS pathologies (2). In a recent 30-d bed rest head-down tilt study with 0.5% CO2 exposure, 5 of 11 subjects developed optic disc edema (4) and the same SHMT1 C1420T and MTRR A66G genetic variants were associated with a larger increase in total retina thickness, a quantitative measure of optic disc edema (5). We published a multi-hit hypothesis of how genetics represents an indispensable element of SANS, which is a multifactorial problem (6). In brief: endothelial dysfunction secondary to genetic, biochemical/nutritional, and physiological (e.g., cardiovascular/fluid shift) f actors could lead to optic disc edema and the other ocular changes that occur in SANS. We expanded this hypothesis to include the possibility that genetic effects on B-vitamin status can alter nitric oxide synthesis and oxidative stress in the endothelium. This in turn could alter the turnover of structural components of the sclera, making it more susceptible to pathologic changes when faced with stressors related to the unrelenting headward fluid shift that occurs in weightlessness and strict head-down tilt bed rest (5). If the relationships that we have observed in flight and ground-based research hold true for the SANS Countermeasure Study, these genetic factors could predict who will be more susceptible to the development of SANS, and more importantly could also provide countermeasure options. As has been shown extensively in the literature, vitamin supplementation can serve to overcome genetic hindrances to one-carbon biochemistry and vascular physiology. Thus, based on our findings, publications, and hypotheses, supported by extensive supporting literature, we had hoped to test in the SANS Countermeasure Study the efficacy of a bioactive B-vitamin complex as a countermeasure to optimize function of the one-carbon pathway and prevent or mitigate optic disc edema during strict 6-degree head down tilt bed rest, and ultimately in space f light. While the supplement is no longer planned to be tested in the SANS Countermeasure Study because of laws regulating supplementation studies like this costing much more than initially planned, we will assess the contribution of one-carbon pathway genetics and B-vitamin status to SANS risk.

S R Zwart↗

Genome-wide Selective Sweeps in Natural Bacterial Populations Revealed by Time-series Metagenomics

Multiple evolutionary models have been proposed to explain the formation of genetically and ecologically distinct bacterial groups. Time-series metagenomics enables direct observation of evolutionary processes in natural populations, and if applied over a sufficiently long time frame, this approach could capture events such as gene-specific or genome-wide selective sweeps. Direct observations of either process could help resolve how distinct groups form in natural microbial assemblages. Here, from a three-year metagenomic study of a freshwater lake, we explore changes in single nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in populations of Chlorobiaceae and Methylophilaceae. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied considerably among closely related, co-occurring Methylophilaceae populations. SNP allele frequencies, as well as the relative abundance of certain genes, changed dramatically over time in each population. Interestingly, SNP diversity was purged at nearly every genome position in one of the Chlorobiaceae populations over the course of three years, while at the same time multiple genes either swept through or were swept from this population. These patterns were consistent with a genome-wide selective sweep, a process predicted by the ‘ecotype model’ of diversification, but not previously observed in natural populations.

Chan, Leong-Keat↗