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Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

ABSTRACT: Genome-wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro-Tom genome were identified by a whole-genome shotgun sequencing analysis to estimate the spectrum and distribution of whole-genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired-end reads for four EMS-induced mutants and three gamma-ray-irradiated lines as well as a wild-type line were obtained by next-generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma-ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1,140,687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild-type Micro-Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild-type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse-genetic approaches.

SUBMITTER: Shirasawa K

PROVIDER: S-EPMC5023996 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

Shirasawa Kenta K Hirakawa Hideki H Nunome Tsukasa T Tabata Satoshi S Isobe Sachiko S

Plant biotechnology journal 20150216 1

Genome-wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro-Tom genome were identified by a whole-genome shotgun sequencing analysis to estimate the spectrum and distribution of whole-genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired-end reads for four EMS-induced mutants and three gamma-ray-irradiated lines as well as a wild-type line were obtained by next-generation sequencing technology. Using bioinformati ...[more]

PMID: 25689669

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Project description:Physical mutagens, such as carbon-ion beams (CIBs) and gamma rays (GRs), induce mutations with high frequency at a relatively low dose and are more user-friendly and environment-friendly in mutation breeding. Previous studies showed that CIBs induced large sized deletions and insertions, and chromosomal rearrangements, whereas GRs induce shorter deletions and insertions, and more frequent base substitutions. However, the difference on the genomic level between CIB- and GR-induced mutations remains to be clarified. In the present study, we re-sequence six mutagenized lines derived from CIB irradiation and four mutagenized lines derived from GRs. A total of 283 and 381 variations are induced in these mutants by CIBs and GRs, respectively, including single base substitutions (SBSs), small insertion and deletions (InDels), multiple nucleotide variants (MNVs). SBSs are the most abundant type of mutation and single base transition is the main form for SBSs. CIB-induced InDels accounted for 25.44% of the total variations, while GR-induced InDels accounted for 17.85%. On the contrary, the frequency of MNVs induced by GRs was approximately 2.19 times that induced by CIBs, which indicates CIBs induced increased InDels, whereas GRs induced increased MNVs. Notably, multiple base deletions (≥5 bp) were induced at a much higher frequency by CIBs than by GRs. We also find mutations induced by CIBs and GRs are unevenly distributed on chromosomes. Unusual high-frequency (HF) and low-frequency (LF) mutation regions are discovered by analyzing mutations per 1Mb along the genome. The mutation frequency within the HF regions were significantly higher than the LF regions (P < 0.05). A large majority of SBSs, InDels, and MNVs induced by CIBs and GRs occurred in upstream and downstream regions. Our study compares difference of mutation profiles induced by the CIB irradiation and GR on rice genomes, and give some clues for understanding the mutagenesis mechanism of physical radiation and improving the mutagenesis efficiency.

Project description:Genetic variations are an important source of germplasm diversity, as it provides an allele resource that contributes to the development of new traits for plant breeding. Gamma rays have been widely used as a physical agent for mutation creation in plants, and their mutagenic effect has attracted extensive attention. However, few studies are available on the comprehensive mutation profile at both the large-scale phenotype mutation screening and whole-genome mutation scanning. In this study, biological effects on M1 generation, large-scale phenotype screening in M2 generation, as well as whole-genome re-sequencing of seven M3 phenotype-visible lines were carried out to comprehensively evaluate the mutagenic effects of gamma rays on Arabidopsis thaliana. A total of 417 plants with visible mutated phenotypes were isolated from 20,502 M2 plants, and the phenotypic mutation frequency of gamma rays was 2.03% in Arabidopsis thaliana. On average, there were 21.57 single-base substitutions (SBSs) and 11.57 small insertions and deletions (InDels) in each line. Single-base InDels accounts for 66.7% of the small InDels. The genomic mutation frequency was 2.78 × 10-10/bp/Gy. The ratio of transition/transversion was 1.60, and 64.28% of the C > T events exhibited the pyrimidine dinucleotide sequence; 69.14% of the small InDels were located in the sequence with 1 to 4 bp terminal microhomology that was used for DNA end rejoining, while SBSs were less dependent on terminal microhomology. Nine genes, on average, were predicted to suffer from functional alteration in each re-sequenced line. This indicated that a suitable mutation gene density was an advantage of gamma rays when trying to improve elite materials for one certain or a few traits. These results will aid the full understanding of the mutagenic effects and mechanisms of gamma rays and provide a basis for suitable mutagen selection and parameter design, which can further facilitate the development of more controlled mutagenesis methods for plant mutation breeding.

Project description: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.

Dataset Information

Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

Publications

Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

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