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Assessing the effect of spaceflight stress on DNA sequence mutation using RNA-sequencing data Cover

Assessing the effect of spaceflight stress on DNA sequence mutation using RNA-sequencing data

Gravitational and Space Research
Volume 13 (2025): Issue 1 (January 2025)
By: Montana S. Knight,  Colleen J. Doherty and  Dahlia M. Nielsen  
Open Access
|Dec 2025

Figures & Tables

Figure 1.

Number of de novo variants identified. Each point represents one RNA-Seq sample.
Number of de novo variants identified. Each point represents one RNA-Seq sample.

Figure. 2.

Proportion of transversion mutations. For each sample, the proportion of de novo variants identified that are transversions is shown. Each point represents one RNA-Seq sample.
Proportion of transversion mutations. For each sample, the proportion of de novo variants identified that are transversions is shown. Each point represents one RNA-Seq sample.

Supplemental Figure S1.

Principal Component Analysis on normalized gene expression counts. The x-axis in each plot reflects variance across Principal Component 1 (PC1), which explained 93.96% of the variance. The y-axis in each reflects Principal Component 2 (PC2), which explained 2.15% of the variance. (A) The PCA plot colored by treatment group: TR=transgenic, WT=wildtype, SF=spaceflight, and GC=ground control. (B) The same PCA plot colored by tissue type. Tissue type appears to account for the majority of the variance in the first principal component. (C) The PCA plot colored by spaceflight condition, which does not appear to explain the variance on these two PCs. (D) The PCA plot colored by genotype, which also does not appear to explain the variance on these two PCs.
Principal Component Analysis on normalized gene expression counts. The x-axis in each plot reflects variance across Principal Component 1 (PC1), which explained 93.96% of the variance. The y-axis in each reflects Principal Component 2 (PC2), which explained 2.15% of the variance. (A) The PCA plot colored by treatment group: TR=transgenic, WT=wildtype, SF=spaceflight, and GC=ground control. (B) The same PCA plot colored by tissue type. Tissue type appears to account for the majority of the variance in the first principal component. (C) The PCA plot colored by spaceflight condition, which does not appear to explain the variance on these two PCs. (D) The PCA plot colored by genotype, which also does not appear to explain the variance on these two PCs.

ANOVA results, testing for the proportion of transversion mutations_

ComparisonDifference EstimateSEP-value
Shoot vs. root tissue0.06240.0072.65E-19
Interaction effect b/w tissue and gravity condition−0.13240.05580.018
Interaction b/w tissue type and non-microgravity spaceflight stress0.14610.06340.021
Interaction b/w gravity and non-microgravity spaceflight stress−3.83920.0735<0.001
Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress−0.36080.07359.03E-07
Spaceflight vs. ground control0.01630.00790.04
Spaceflight with gravity control vs. ground control0.01770.00890.048
Spaceflight with gravity control vs. ground control in shoot tissue0.0310.00787.81E-05
Spaceflight vs. ground control in shoot tissue0.03450.00694.72E-07

Final numbers of variants for each sample_

SampleLineTissue TypeTreatmentResponse
FLS18Wild-TypeShootSpaceflight with Gravity Control1568
FLS28Wild-TypeShootNormal Spaceflight1802
FLS38Wild-TypeShootNormal Spaceflight1688
FLS48Wild-TypeShootSpaceflight with Gravity Control1668
FLS58Wild-TypeShootSpaceflight with Gravity Control1474
FLS68Wild-TypeShootNormal Spaceflight2106
FLS78Wild-TypeShootNormal Spaceflight1966
GCS1Wild-TypeShootGround Control1209
GCS14Wild-TypeShootGround Control1607
GCS18Wild-TypeShootGround Control622
FL08Wild-TypeRootSpaceflight with Gravity Control617
FL18Wild-TypeRootSpaceflight with Gravity Control522
FL28Wild-TypeRootNormal Spaceflight500
FL38Wild-TypeRootNormal Spaceflight511
FL48Wild-TypeRootSpaceflight with Gravity Control414
FL58Wild-TypeRootSpaceflight with Gravity Control471
FL68Wild-TypeRootNormal Spaceflight454
FL78Wild-TypeRootNormal Spaceflight704
GC14Wild-TypeRootGround Control573
GC16Wild-TypeRootGround Control866
GCS13TransgenicShootGround Control1552
GCS17TransgenicShootGround Control754
GCS2TransgenicShootGround Control1094
FLS23TransgenicShootNormal Spaceflight1628
FLS33TransgenicShootNormal Spaceflight1644
FLS63TransgenicShootNormal Spaceflight1688
FLS73TransgenicShootNormal Spaceflight1946
FLS13TransgenicShootSpaceflight with Gravity Control1939
FLS43TransgenicShootSpaceflight with Gravity Control1683
FLS53TransgenicShootSpaceflight with Gravity Control2189
GC01TransgenicRootGround Control904
GC13TransgenicRootGround Control473
FL23TransgenicRootNormal Spaceflight518
FL33TransgenicRootNormal Spaceflight515
FL63TransgenicRootNormal Spaceflight567
FL73TransgenicRootNormal Spaceflight498
FL03TransgenicRootSpaceflight with Gravity Control404
FL13TransgenicRootSpaceflight with Gravity Control603
FL43TransgenicRootSpaceflight with Gravity Control452
FL53TransgenicRootSpaceflight with Gravity Control464

Least-squared means from Analysis of Variance_

GenotypeTissue TypeTreatmentLSMean EstimateStandard Error
Wild-TypeShootGround Control1146126.55
Wild-TypeShootSpaceflight with Gravity Control1570126.55
Wild-TypeShootSpaceflight1890.5109.59
Wild-TypeRootGround Control719.5154.99
Wild-TypeRootSpaceflight with Gravity Control506109.59
Wild-TypeRootSpaceflight542.25109.59
TransgenicShootGround Control1133.33126.55
TransgenicShootSpaceflight with Gravity Control1937126.55
TransgenicShootSpaceflight1726.5109.59
TransgenicRootGround Control688.5154.99
TransgenicRootSpaceflight with Gravity Control480.75109.59
TransgenicRootSpaceflight524.5109.59

Number of variants remaining after each filtering step_

Called by Mutect2Hard FilteredDeduplicatedDifficult to Sequence Reads RemovedFiltered by Gene Expression
Transgenic Shoot4803726220.13097.61639.71611.7
Transgenic Root14638.11473.3721.8549539.8
Wild-type Shoot49944.725162.52208.21589.21571
Wild-type Root15148.51516.4745.7571.5563.2

ANOVA results, testing for differences in numbers of de novo mutations_

ComparisonDifference EstimateSEP-value
Shoot vs. root tissue990.30671.5231.34E-43
Interaction effect b/w tissue and gravity condition−1708.917572.9590.003
Interaction b/w tissue type and non-microgravity spaceflight stress3327.833651.4323.25E-07
Interaction b/w gravity and non-microgravity spaceflight stress−12248.417753.9812.42E-59
Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress−4322.917753.9819.84E-09
Gravity vs. microgravity148.30271.620.038
Spaceflight vs. ground control225.35481.4290.006
Spaceflight with gravity control vs. ground control201.60492.2350.029
Spaceflight with gravity control vs. ground control in shoot tissue613.833126.5451.23E-06
Spaceflight vs. ground control in shoot tissue641.333107.2842.26E-09

Number of variants identified per genotype/tissue type combination_

Number of Likely Shared Variants from Mutect2Number of Joint Genotyped Variants from HaplotypeCallerNumber of Variants in the Ultimate PON
Transgenic Shoot10933132917469
Transgenic Root683595056954
Wild Type Shoot11408139028021
Wild Type Root681895106943

Analysis of Variance results_

ComparisonDifference EstimateSEP-value
Shoot vs. root tissue−990.30671.5231.34E-43
Wild-type vs. transgenic genotype19.38971.5230.786
Interaction effect b/w tissue and genotype−264.333429.1360.538
Interaction effect b/w tissue and gravity condition−1708.917572.9590.003
Interaction b/w tissue type and non-microgravity spaceflight stress3327.833651.4323.25E-07
Interaction b/w A. thaliana genotype and gravity condition−661.583572.9590.248
Interaction b/w A. thaliana genotype and non-microgravity spaceflight stress−247.333651.4320.704
Interaction b/w gravity and non-microgravity spaceflight stress−12248.417753.9812.42E-59
Interaction b/w tissue type, A. thaliana genotype, and gravity condition−703.083572.9590.22
Interaction b/w tissue type, A. thaliana genotype, and non-microgravity spaceflight stress−209.333651.4320.748
Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress−4322.917753.9819.84E-09
Interaction b/w A. thaliana genotype, gravity condition, and non-microgravity spaceflight stress−792.583753.9810.293
Interaction b/w tissue type, A. thaliana genotype, gravity condition, and non-microgravity spaceflight stress−648.083753.9810.39
Gravity vs. microgravity148.30271.620.038
Gravity vs. microgravity in space47.580.6570.556
Gravity vs. microgravity in space in shoot tissue55118.3720.642
Gravity vs. microgravity in space in root tissue40109.5920.715
Spaceflight vs. ground control225.35481.4290.006
Spaceflight with gravity control vs. ground control−201.60492.2350.029
Spaceflight with gravity control vs. ground control in shoot tissue−613.833126.5451.23E-06
Spaceflight with gravity control vs. ground control in root tissue210.625134.2220.117
Spaceflight vs. ground control in shoot tissue641.333107.2842.26E-09
Spaceflight vs. ground control in root tissue−190.625122.5270.12

Number of samples for each treatment combination_

GenotypeTissue TypeSpace Flight μGSpace Flight 0.76G by centrifugationGround Control 1G on EarthTotal
Wild TypeRoot44210
Wild TypeShoot43310
InsP 5-ptase TransgenicRoot44210
InsP 5-ptase TransgenicShoot43310
DOI: https://doi.org/10.2478/gsr-2025-0009 | Journal eISSN: 2332-7774
Journal RSS Feed
Language: English
Page range: 121 - 134
Published on: Dec 12, 2025
Published by: American Society for Gravitational and Space Research
In partnership with: Paradigm Publishing Services
Publication frequency: 2 issues per year
Keywords:
Space flight,
mutation rates,
space radiation,
microgravity,
DNA,
RNA-Seq
Related subjects:
Life sciences,
Life sciences, other,
Materials sciences,
Materials sciences, other,
Physics,
Physics, other

© 2025 Montana S. Knight, Colleen J. Doherty, Dahlia M. Nielsen, published by American Society for Gravitational and Space Research
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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