Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice

Abstract

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a distinct mechanism from those of chemical mutagens or gamma rays.We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three earlyheading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10 -7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a highimpact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

Evaluation of Toraja (Indonesia) local aromatic rice mutant developed using heavy-ion beam irradiation

“…PB-A produced 5 dwarf phenotype plants with a frequency of 2.6% and 79 semi-dwarf plants with a frequency of 40.3%, while PB-C and the control did not produce dwarf plants (Table 1). Dwarf rice plants caused by ion beam, as observed in the present study was previously reported by Oono et al (2020). Zheng et al (2020) showed that heavy-ion beam irradiation was effective in inducing mutations in rice, and revealed the characteristics of mutations at the genome level.…”

Frequency and Spectrum of Mutations Induced by Gamma Rays Revealed by Phenotype Screening and Whole-Genome Re-Sequencing in Arabidopsis thaliana

“…The molecular mutation profile at the genome scale can also be used for selecting a proper mutagen for mutation breeding and gene function analysis. Re-sequencing analysis of progeny of mutagenized plant material has provided a new perspective aimed at characterizing the mutation molecular profile of various physical mutagens at the whole-genome scale in the last 10 years [ 31 , 33 , 36 , 40 , 41 , 47 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. In earlier research, only one mutant line was sequenced since it was still costly to perform NGS analysis.…”

“…To date, there is no definite standard regarding which line and generation should be used for re-sequencing. Some studies have used randomly selected M 2 individuals [ 31 , 40 , 64 ]; some have preferred higher-generation plants displaying visibly mutated phenotypes [ 35 , 41 , 55 , 58 , 65 ]. Few studies were performed in the M 1 generation, as M 1 plants are genetic chimeras.…”

“…The ratio value was 1.0, 0.89, 0.99, and 0.88 for the fast neutron (60 Gy), argon ions (290 keV/μm, 50Gy), and carbon ions (30.0 keV/μm, 400Gy; 50.0 keV/μm, 200Gy; 107 keV/μm, 175Gy) irradiation, respectively [ 40 , 58 , 61 , 65 , 69 ]. In rice, the Ts/Tv was 0.90 in carbon ions (135 MeV/u, 30 keV/μm), 2.4 for carbon ions (25.9 MeV/u, 76 keV/μm, 40 Gy), 1.4 for fast neutron (20 Gy), 1.49–1.6 for gamma rays (250Gy) [ 35 , 41 , 55 , 64 , 70 ]. In tomato, the ratio was 0.86 for gamma rays (300Gy).…”

Responsible Gene Analysis of Phenotypic Mutants Revealed the Linear Energy Transfer (LET)-Dependent Mutation Spectrum in Rice

“…The WES, WGS, and bioinformatics analyses were performed as previously described (Ichida et al 2019, Oono et al 2020, Udagawa et al 2021. Briefly, the extracted DNA was fragmented by sonication and used to construct a sequencing library using the NextFlex Rapid DNA-Seq library prep kit (Bio Scientific, Austin, TX, USA) according to the manufacturerʼs protocol.…”