Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Abstract

SUMMARYHeavy-ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole-genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array-CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar-ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.

Frequent generation of mutants with coincidental changes in multiple traits via ion-beam irradiation in soybean

“…radiation and bring about a large deletion and/or rearrangement of a DNA segment (Shikazono et al, 2005;Tanaka et al, 2010;Hirano et al, 2012). A genome-wide analysis of structural changes induced by ion-beam irradiation in Arabidopsis revealed that deletions as large as 1.2 Mbp could occur (Hirano et al, 2015). Such a large deletion can involve the loss of multiple linked genes and, therefore, may explain some of the coincidental changes observed in this study.…”

AMAP: A pipeline for whole-genome mutation detection in <i>Arabidopsis thaliana</i>

“…The size of deletions increases with increasing value of the linear energy transfers (LETs) of heavy-ion beams (Kazama et al, 2011(Kazama et al, , 2013Hirano et al, 2012). Whole-genome identification using HTS confirmed that base substitutions, deletions, insertions and chromosomal rearrangements were induced at the whole-genome level (Hirano et al, 2015). Thus, it is now possible to determine any type of mutation using HTS.…”

“…In A. thaliana, fast-neutron-induced mutations were revealed to be mainly base substitutions and small deletions (Belfield et al, 2012). We have previously identified the mutation spectrum of the heavy-ion beam in A. thaliana as comprising base substitutions, deletions, insertions and chromosomal rearrangements (Hirano et al, 2015). The size of deletions increases with increasing value of the linear energy transfers (LETs) of heavy-ion beams (Kazama et al, 2011(Kazama et al, , 2013Hirano et al, 2012).…”

“…The possible causes of false positives include mismapping of sequencing reads or SNPs between the original accession and the accession used in mutation induction. Belfield et al (2012) and Hirano et al (2015) confirmed all candidate mutations using a genome browser, but this is a very time-consuming process.…”

“…In AMAP, false positives are automatically determined by searching repetitive sequences near candidate mutations and by comparing lists of candidate mutations in sequenced mutants. We tested AMAP using HTS data that were previously analyzed by Hirano et al (2015). HTS analysis using AMAP will allow the acceleration of forward genetics and gene function analysis.…”