Table 1.
Number of samples for each treatment combination.
| Genotype | Tissue Type | Space Flight μG | Space Flight 0.76G by centrifugation | Ground Control 1G on Earth | Total |
|---|---|---|---|---|---|
| Wild Type | Root | 4 | 4 | 2 | 10 |
| Wild Type | Shoot | 4 | 3 | 3 | 10 |
| InsP 5-ptase Transgenic | Root | 4 | 4 | 2 | 10 |
| InsP 5-ptase Transgenic | Shoot | 4 | 3 | 3 | 10 |
Table 2.
Number of variants identified per genotype/tissue type combination.
| Number of Likely Shared Variants from Mutect2 | Number of Joint Genotyped Variants from HaplotypeCaller | Number of Variants in the Ultimate PON | |
|---|---|---|---|
| Transgenic Shoot | 10933 | 13291 | 7469 |
| Transgenic Root | 6835 | 9505 | 6954 |
| Wild Type Shoot | 11408 | 13902 | 8021 |
| Wild Type Root | 6818 | 9510 | 6943 |
Table 3.
ANOVA results, testing for differences in numbers of de novo mutations.
| Comparison | Difference Estimate | SE | P-value |
|---|---|---|---|
| Shoot vs. root tissue | 990.306 | 71.523 | 1.34E-43 |
| Interaction effect b/w tissue and gravity condition | −1708.917 | 572.959 | 0.003 |
| Interaction b/w tissue type and non-microgravity spaceflight stress | 3327.833 | 651.432 | 3.25E-07 |
| Interaction b/w gravity and non-microgravity spaceflight stress | −12248.417 | 753.981 | 2.42E-59 |
| Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress | −4322.917 | 753.981 | 9.84E-09 |
| Gravity vs. microgravity | 148.302 | 71.62 | 0.038 |
| Spaceflight vs. ground control | 225.354 | 81.429 | 0.006 |
| Spaceflight with gravity control vs. ground control | 201.604 | 92.235 | 0.029 |
| Spaceflight with gravity control vs. ground control in shoot tissue | 613.833 | 126.545 | 1.23E-06 |
| Spaceflight vs. ground control in shoot tissue | 641.333 | 107.284 | 2.26E-09 |
Figure 1.
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.
Table 4.
ANOVA results, testing for the proportion of transversion mutations.
| Comparison | Difference Estimate | SE | P-value |
|---|---|---|---|
| Shoot vs. root tissue | 0.0624 | 0.007 | 2.65E-19 |
| Interaction effect b/w tissue and gravity condition | −0.1324 | 0.0558 | 0.018 |
| Interaction b/w tissue type and non-microgravity spaceflight stress | 0.1461 | 0.0634 | 0.021 |
| Interaction b/w gravity and non-microgravity spaceflight stress | −3.8392 | 0.0735 | <0.001 |
| Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress | −0.3608 | 0.0735 | 9.03E-07 |
| Spaceflight vs. ground control | 0.0163 | 0.0079 | 0.04 |
| Spaceflight with gravity control vs. ground control | 0.0177 | 0.0089 | 0.048 |
| Spaceflight with gravity control vs. ground control in shoot tissue | 0.031 | 0.0078 | 7.81E-05 |
| Spaceflight vs. ground control in shoot tissue | 0.0345 | 0.0069 | 4.72E-07 |
Supplemental Table S1.
Number of variants remaining after each filtering step.
| Called by Mutect2 | Hard Filtered | Deduplicated | Difficult to Sequence Reads Removed | Filtered by Gene Expression | |
|---|---|---|---|---|---|
| Transgenic Shoot | 48037 | 26220.1 | 3097.6 | 1639.7 | 1611.7 |
| Transgenic Root | 14638.1 | 1473.3 | 721.8 | 549 | 539.8 |
| Wild-type Shoot | 49944.7 | 25162.5 | 2208.2 | 1589.2 | 1571 |
| Wild-type Root | 15148.5 | 1516.4 | 745.7 | 571.5 | 563.2 |
Supplemental Table S2.
Final numbers of variants for each sample.
| Sample | Line | Tissue Type | Treatment | Response |
|---|---|---|---|---|
| FLS18 | Wild-Type | Shoot | Spaceflight with Gravity Control | 1568 |
| FLS28 | Wild-Type | Shoot | Normal Spaceflight | 1802 |
| FLS38 | Wild-Type | Shoot | Normal Spaceflight | 1688 |
| FLS48 | Wild-Type | Shoot | Spaceflight with Gravity Control | 1668 |
| FLS58 | Wild-Type | Shoot | Spaceflight with Gravity Control | 1474 |
| FLS68 | Wild-Type | Shoot | Normal Spaceflight | 2106 |
| FLS78 | Wild-Type | Shoot | Normal Spaceflight | 1966 |
| GCS1 | Wild-Type | Shoot | Ground Control | 1209 |
| GCS14 | Wild-Type | Shoot | Ground Control | 1607 |
| GCS18 | Wild-Type | Shoot | Ground Control | 622 |
| FL08 | Wild-Type | Root | Spaceflight with Gravity Control | 617 |
| FL18 | Wild-Type | Root | Spaceflight with Gravity Control | 522 |
| FL28 | Wild-Type | Root | Normal Spaceflight | 500 |
| FL38 | Wild-Type | Root | Normal Spaceflight | 511 |
| FL48 | Wild-Type | Root | Spaceflight with Gravity Control | 414 |
| FL58 | Wild-Type | Root | Spaceflight with Gravity Control | 471 |
| FL68 | Wild-Type | Root | Normal Spaceflight | 454 |
| FL78 | Wild-Type | Root | Normal Spaceflight | 704 |
| GC14 | Wild-Type | Root | Ground Control | 573 |
| GC16 | Wild-Type | Root | Ground Control | 866 |
| GCS13 | Transgenic | Shoot | Ground Control | 1552 |
| GCS17 | Transgenic | Shoot | Ground Control | 754 |
| GCS2 | Transgenic | Shoot | Ground Control | 1094 |
| FLS23 | Transgenic | Shoot | Normal Spaceflight | 1628 |
| FLS33 | Transgenic | Shoot | Normal Spaceflight | 1644 |
| FLS63 | Transgenic | Shoot | Normal Spaceflight | 1688 |
| FLS73 | Transgenic | Shoot | Normal Spaceflight | 1946 |
| FLS13 | Transgenic | Shoot | Spaceflight with Gravity Control | 1939 |
| FLS43 | Transgenic | Shoot | Spaceflight with Gravity Control | 1683 |
| FLS53 | Transgenic | Shoot | Spaceflight with Gravity Control | 2189 |
| GC01 | Transgenic | Root | Ground Control | 904 |
| GC13 | Transgenic | Root | Ground Control | 473 |
| FL23 | Transgenic | Root | Normal Spaceflight | 518 |
| FL33 | Transgenic | Root | Normal Spaceflight | 515 |
| FL63 | Transgenic | Root | Normal Spaceflight | 567 |
| FL73 | Transgenic | Root | Normal Spaceflight | 498 |
| FL03 | Transgenic | Root | Spaceflight with Gravity Control | 404 |
| FL13 | Transgenic | Root | Spaceflight with Gravity Control | 603 |
| FL43 | Transgenic | Root | Spaceflight with Gravity Control | 452 |
| FL53 | Transgenic | Root | Spaceflight with Gravity Control | 464 |
Supplemental Table S3.
Least-squared means from Analysis of Variance.
| Genotype | Tissue Type | Treatment | LSMean Estimate | Standard Error |
|---|---|---|---|---|
| Wild-Type | Shoot | Ground Control | 1146 | 126.55 |
| Wild-Type | Shoot | Spaceflight with Gravity Control | 1570 | 126.55 |
| Wild-Type | Shoot | Spaceflight | 1890.5 | 109.59 |
| Wild-Type | Root | Ground Control | 719.5 | 154.99 |
| Wild-Type | Root | Spaceflight with Gravity Control | 506 | 109.59 |
| Wild-Type | Root | Spaceflight | 542.25 | 109.59 |
| Transgenic | Shoot | Ground Control | 1133.33 | 126.55 |
| Transgenic | Shoot | Spaceflight with Gravity Control | 1937 | 126.55 |
| Transgenic | Shoot | Spaceflight | 1726.5 | 109.59 |
| Transgenic | Root | Ground Control | 688.5 | 154.99 |
| Transgenic | Root | Spaceflight with Gravity Control | 480.75 | 109.59 |
| Transgenic | Root | Spaceflight | 524.5 | 109.59 |
Supplemental Table S4.
Analysis of Variance results.
| Comparison | Difference Estimate | SE | P-value |
|---|---|---|---|
| Shoot vs. root tissue | −990.306 | 71.523 | 1.34E-43 |
| Wild-type vs. transgenic genotype | 19.389 | 71.523 | 0.786 |
| Interaction effect b/w tissue and genotype | −264.333 | 429.136 | 0.538 |
| Interaction effect b/w tissue and gravity condition | −1708.917 | 572.959 | 0.003 |
| Interaction b/w tissue type and non-microgravity spaceflight stress | 3327.833 | 651.432 | 3.25E-07 |
| Interaction b/w A. thaliana genotype and gravity condition | −661.583 | 572.959 | 0.248 |
| Interaction b/w A. thaliana genotype and non-microgravity spaceflight stress | −247.333 | 651.432 | 0.704 |
| Interaction b/w gravity and non-microgravity spaceflight stress | −12248.417 | 753.981 | 2.42E-59 |
| Interaction b/w tissue type, A. thaliana genotype, and gravity condition | −703.083 | 572.959 | 0.22 |
| Interaction b/w tissue type, A. thaliana genotype, and non-microgravity spaceflight stress | −209.333 | 651.432 | 0.748 |
| Interaction b/w tissue type, gravity condition, and non-microgravity spaceflight stress | −4322.917 | 753.981 | 9.84E-09 |
| Interaction b/w A. thaliana genotype, gravity condition, and non-microgravity spaceflight stress | −792.583 | 753.981 | 0.293 |
| Interaction b/w tissue type, A. thaliana genotype, gravity condition, and non-microgravity spaceflight stress | −648.083 | 753.981 | 0.39 |
| Gravity vs. microgravity | 148.302 | 71.62 | 0.038 |
| Gravity vs. microgravity in space | 47.5 | 80.657 | 0.556 |
| Gravity vs. microgravity in space in shoot tissue | 55 | 118.372 | 0.642 |
| Gravity vs. microgravity in space in root tissue | 40 | 109.592 | 0.715 |
| Spaceflight vs. ground control | 225.354 | 81.429 | 0.006 |
| Spaceflight with gravity control vs. ground control | −201.604 | 92.235 | 0.029 |
| Spaceflight with gravity control vs. ground control in shoot tissue | −613.833 | 126.545 | 1.23E-06 |
| Spaceflight with gravity control vs. ground control in root tissue | 210.625 | 134.222 | 0.117 |
| Spaceflight vs. ground control in shoot tissue | 641.333 | 107.284 | 2.26E-09 |
| Spaceflight vs. ground control in root tissue | −190.625 | 122.527 | 0.12 |
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.