Mutational effects of ubiquitously present gamma radiation on Arabidopsis thaliana: insight into radiosensitivity in the reproductive stage

Earth has always been exposed to ionizing radiation from natural sources, and man-made sources have added to this radiation. In order to assess mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life-cycle of Arabidopsis thaliana under controlled conditions. Resequencing data from 12 M1 lines and 36 M2 progeny derived under gamma-irradiation conditions ranging from 0.0 to 2.0 Gy/d were obtained to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and a low-to-middling dose of radiation was assessed by statistical modeling. The increasing of de novo mutations in response to doses of irradiation fit the negative binomial model, accounting for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, with deletions being more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation are more intensive during the reproductive stage. These outcomes could provide valuable insights into practical strategies for environmental radioprotection of plants on Earth and in space.


INTRODUCTION
Since the discovery of Muller (1927) [1] and Stadler (1928) [2] that ionizing radiation 47 (X-rays) induces mutations, the biological effects of ionizing radiation have been 48 extensively studied in the field of genetics. In the last nearly one hundred years, the ubiquitously present radiation, as their chronic irradiation was performed on plants in 72 the vegetative growth stage (approximately two weeks before bolting), but not in the 73 reproductive stage. Generally, the reproductive growth stage is more radiosensitive than 74 the vegetative stage, although life stage-specific sensitivity to mutations has not been 75 explored on a genome-wide scale. In this study, therefore, we performed a whole-76 genome resequencing analysis of mutations induced by life cycle-specific chronic 77 irradiation in A. thaliana. 78 Earth has always been exposed to ionizing radiation from natural sources, and man-79 made sources have added to this radiation. Absorbed dose rates in the air of terrestrial 80 gamma radiation typically range from 10 to 200 nGy/h over 55 countries worldwide 81 [16]. Another field where the effects of ubiquitously present radiation are important is 82 space, where irradiation dose rates are more than a hundred times higher than in 83 terrestrial environments. For example, the radiation dose at the International Space 84 Station (ISS) can vary, but is estimated to be on average 0.327 mGy/d [17]. From the 85 perspective of radioprotection, a 'low dose' of radiation from any man-made sources 86 has been defined as 100 mGy or less of sparsely ionizing radiation, and a 'low dose-87 rate' as less than 0. Additionally, as mentioned above, the mutational effect of ionizing radiation at very low 96 dose rates (~1 mGy/h) has not been determined in plant models [14]. Thus, the 97 mutational effect of 'low doses' and/or 'low dose-rates' above the natural background 98 radiation are difficult to reliably distinguish between in terms of low risk or zero risk. In 99 this study, therefore, we used low-to-moderate doses of gamma radiation (less than 2 100 Gy/d) to more clearly identify the mutational effects of chronic radiation throughout the 101 life-cycle of plants. 102 Mutations are intrinsically difficult to study because de novo mutation events are very 103 rare, even under exposure to ionizing radiation. In order to overcome the rarity of the M2 generation. Therefore, in this study, we applied the latter approach to identify de 122 novo mutations in M2 generation to test the hypothesis that radiation-induced mutations 123 more frequently occur in the reproductive stage than in the vegetative stage.

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The main objective of the present study was to assess the mutation frequency and

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Plant materials, gamma irradiation, and whole-genome sequencing 136 Detail information of the irradiation experiment is provided in Supplemental Text S1.      Table S1). After removing the low quality, 252 unpaired, and duplicate reads, more than 99.5% of clean reads were mapped to the 253 TAIR10 reference genome. The average depth of coverage was 65X and 98.4% of the 254 genome on average had at least 10X coverage.  Table S4). Thereafter, all of the 586 mutation sites were used for 265 downstream analyses.

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The effect of gamma irradiation on mutations 268 Mutation frequency and mutation rate increased approximately 5-to 14-fold with an 269 increase in the irradiation dose from 0.0 to 2.0 Gy/d ( Table 1). The best-fitting statistical    Table S2). The best-fitting statistical model for the relationship between the number of high-impact mutations and irradiation dose showed 310 a significant increasing effect of irradiation (Supplementary Figure S3 and Table S8).

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The same treads in radiation response were also observed for the number of moderate-, zygote development, and then seed maturation. Therefore, we concluded that mutational 369 radiosensitivity is higher in the reproductive stage, especially for more irradiated plants. 370 From the other point of view, family-shared mutations (i.e., multiple common 371 mutations within a full-sib family) were interpreted as evidence for mutation events that 372 occurred prior to gametogenesis. According to the lows of Mendelian inheritance, if de 373 novo germline mutations were occurred in the vegetative growth stage, 84.4% of these 374 mutations would be transmitted to more than two out of the three full-sib M2 progeny 375 and then assigned as family-shared mutations in this study system. However, the 376 observed percentages of family-shared mutations in total mutations were low for the 377 irradiated progeny (6.3-11.8%: Table 1 and Supplementary Table S2). In a strict sense, 378 family-shared mutations were derived from mutation events not only in the vegetative 379 growth stage but also in the early reproductive stage, after bolting until gametogenesis.