Project "Ion Beam Mutagenesis", Department of Radiation-Applied Biology Research, Takasaki Advanced Radiation Research Institute, National Institutes for Quantum Science and Technology, Takasaki, Japan.
PLoS Genet. 2022 Jan 20;18(1):e1009979. doi: 10.1371/journal.pgen.1009979. eCollection 2022 Jan.
Radiation-induced mutations have been detected by whole-genome sequencing analyses of self-pollinated generations of mutagenized plants. However, large DNA alterations and mutations in non-germline cells were likely missed. In this study, in order to detect various types of mutations in mutagenized M1 plants, anthocyanin pigmentation was used as a visible marker of mutations. Arabidopsis seeds heterozygous for the anthocyanin biosynthetic genes were irradiated with gamma-rays. Anthocyanin-less vegetative sectors resulting from a loss of heterozygosity were isolated from the gamma-irradiated M1 plants. The whole-genome sequencing analysis of the sectors detected various mutations, including structural variations (SVs) and large deletions (≥100 bp), both of which have been less characterized in the previous researches using gamma-irradiated plant genomes of M2 or later generations. Various types of rejoined sites were found in SVs, including no-insertion/deletion (indel) sites, only-deletion sites, only-insertion sites, and indel sites, but the rejoined sites with 0-5 bp indels represented most of the SVs. Examinations of the junctions of rearrangements (SVs and large deletions), medium deletions (10-99 bp), and small deletions (2-9 bp) revealed unique features (i.e., frequency of insertions and microhomology) at the rejoined sites. These results suggest that they were formed preferentially via different processes. Additionally, mutations that occurred in putative single M1 cells were identified according to the distribution of their allele frequency. The estimated mutation frequencies and spectra of the M1 cells were similar to those of previously analyzed M2 cells, with the exception of the greater proportion of rearrangements in the M1 cells. These findings suggest there are no major differences in the small mutations (<100 bp) between vegetative and germline cells. Thus, this study generated valuable information that may help clarify the nature of gamma-irradiation-induced mutations and their occurrence in cells that develop into vegetative or reproductive tissues.
通过对诱变植物自花授粉世代的全基因组测序分析，已经检测到辐射诱导的突变。然而，非生殖细胞中的大 DNA 改变和突变可能被遗漏。在这项研究中，为了检测诱变 M1 植物中的各种类型的突变，将花色素苷着色用作突变的可见标记。用γ射线照射含有花色素苷生物合成基因的拟南芥种子。从γ辐照的 M1 植物中分离出由于杂合性丢失而导致的无花色素苷营养组织。对组织的全基因组测序分析检测到各种突变，包括结构变异（SV）和大片段缺失（≥100bp），这两种突变在前几代使用γ辐照的 M2 或更晚代植物基因组的研究中较少被描述。在 SV 中发现了各种类型的重连位点，包括无插入/缺失（indel）位点、仅缺失位点、仅插入位点和 indel 位点，但 0-5bp indel 的重连位点代表了大多数 SV。对重排（SV 和大片段缺失）、中等缺失（10-99bp）和小缺失（2-9bp）的连接处的检查揭示了重连位点的独特特征（即插入和微同源性的频率）。这些结果表明它们是通过不同的过程优先形成的。此外，根据等位基因频率的分布，鉴定了发生在假定的单个 M1 细胞中的突变。M1 细胞的突变频率和谱与之前分析的 M2 细胞相似，除了 M1 细胞中重排的比例更大。这些发现表明，生殖细胞和营养细胞之间的小突变（<100bp）没有重大差异。因此，这项研究提供了有价值的信息，可能有助于阐明γ辐射诱导的突变的性质及其在发育为营养组织或生殖组织的细胞中的发生。
