Heavy-ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double-stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV μm ) and C ions (LET: 30.0 keV μm ), by whole-genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar- and C-ion irradiation, respectively. Conversely, C ions induced more single-base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar- and C-ion irradiation, respectively. Moreover, the rearrangements induced by Ar-ion irradiation were more complex than those induced by C-ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high-throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements.

Detection of mutations at the whole-genome level is now possible by the use of high-throughput sequencing. However, determining mutations is a time-consuming process due to the number of false positives provided by mutation-detecting programs. AMAP (automated mutation analysis pipeline) was developed to overcome this issue. AMAP integrates a set of well-validated programs for mapping (BWA), removal of potential PCR duplicates (Picard), realignment (GATK) and detection of mutations (SAMtools, GATK, Pindel, BreakDancer and CNVnator). Thus, all types of mutations such as base substitution, deletion, insertion, translocation and chromosomal rearrangement can be detected by AMAP. In addition, AMAP automatically distinguishes false positives by comparing lists of candidate mutations in sequenced mutants. We tested AMAP by inputting already analyzed read data derived from three individual Arabidopsis thaliana mutants and confirmed that all true mutations were included in the list of candidate mutations. The result showed that the number of false positives was reduced to 12% of that obtained in a previous analysis that lacked a process of reducing false positives. Thus, AMAP will accelerate not only the analysis of mutation induction by individual mutagens but also the process of forward genetics.

Undaria pinnatifida, the brown macroalga, is a major commercial edible seaweed, and there is increasing interest in breeding new, improved cultivars for market expansion. In this study, we attempted to establish mutagenesis in U. pinnatifida using Ar and C ion beams as mutagens to meet future demands. To screen irradiated generations for mutants, U. pinnatifida zoospores irradiated with Ar and C ion beams were cultivated in plastic Petri dishes. Some gametophytes derived from the irradiated zoospores showed growth arrest or cell death at the initial developmental stage. Although the growth inhibition and lethal effects were observed at high doses of each ion irradiation, the Ar ion irradiation had high biological effects on cell division and growth. The gametophytes that showed a reduction in cell elongation were designated as an inhibited cell elongation mutant. A comparison of the mutant induction frequencies revealed that the C ion beam showed a higher frequency than the Ar ion beam. The highest frequency was 0.83% at 12.5 Gy of the C ion beam. We determined the total number of sporophytes and embryos per female gametophyte after sporophyte induction. High-dose irradiation with the Ar ion beam decreased the embryo and sporophyte formation, suggesting that the Ar ion beam also has exhibited high biological effects on the fertilization or embryogenesis processes or both. The developed heavy ion mutagenesis and mutant screening methods would be useful for mutation breeding and constructing specific mutant libraries in brown algae, and not only in U. pinnatifida.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

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