Yusuke Kazama

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

Heavy-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.

Phenotypic spectrum of Parachlorella kessleri (Chlorophyta) mutants produced by heavy-ion irradiation.

Heavy-ion mutagenesis is a technology used for effective production of genetic mutants. This study demonstrates that algal breeding using a unicellular alga, Parachlorella kessleri, by heavy-ion mutagenesis can improve lipid yield in laboratory experiments. The primary screening yielded 23 mutants among which a secondary screening yielded 7 strains, which were subjected to phenotypic assays. P. kessleri strains produced by heavy-ion radiation spanned a broad spectrum of phenotypes that differed in lipid content and fatty acid profiles. Starch grain morphology was distinctively altered in one of the mutants. The growth of strain PK4 was comparable to that of the wild type under stress-free culture conditions, and the mutant also produced large quantities of lipids, a combination of traits that may be of commercial interest. Thus, heavy-ion irradiation is an effective mutagenic agent for microalgae and may have potential in the production of strains with gain-of-function phenotypes.

A new physical mapping approach refines the sex-determining gene positions on the Silene latifolia Y-chromosome

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving “the travelling salesman problem”, and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement

Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves β-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H+-PPase activity is required for gluconeogenesis. Lack of H+-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi) levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE). Here, we mutagenized fugu5-1 seeds with 12C6+ heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s) involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm), similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2) as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1, and A#3-1; fugu5-1. Taken together, these results suggest that defects in either H+-PPase or ECH2 compromise cell proliferation due to defects in mobilizing seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post-mitotic cell expansion stage of cotyledon development, probably by converting indolebutyric acid to indole acetic acid.

Level of VERNALIZATION 1 expression is correlated with earliness in extra early-flowering mutant wheat lines

Four extra early-flowering mutants, named extra early-flowering1 (exe1), exe2, exe3, and exe4, were identified in Triticum monococcum strain KU104-1 following heavy-ion beam mutagenesis. The four exe mutants fell into two groups, namely Type I (moderately extra early-flowering type; exe1 and exe3) and Type II (extremely extra early-flowering type; exe2 and exe4). Analysis of plant development in a growth chamber showed that the speed of leaf emergence was accelerated in exe mutants at the reproductive stage compared to wild-type (WT) plants. The speed of leaf emergence was faster in Type II than Type I plants. Analysis of VERNALIZATION 1 (VRN1), a flowering promoter gene, showed that it was more highly expressed in seedlings at early developmental stages in Type II mutants than Type I mutants. These findings indicate that the difference in earliness between Type I and Type II mutants is associated with the level of VRN1 expression. The original KU104-1 is an einkorn wheat strain that carries a null allele of the VRN2 gene, a repressor of flowering. Thus, our results indicate that the level of VRN1 expression controls earliness in exe mutants independently of VRN2.

Ion Beam Breeding and Gene Discovery for Function Analyses Using Mutants

The RIKEN RI Beam Factory (RIBF) is the one of the biggest facilities to accelerate heavy ions in the world. Nuclear physics is the primary subject of the RIBF. You may know RIKEN as nuclear physics laboratories. Its activities cover every scientific and technological fields, even in agriculture. We have developed a unique technology for mutation induction by using heavy-ion beams from particle accelerators at the RIBE, through an efficient synergistic link between agricultural science and accelerator physics. At relatively low doses, ion beams induce mutations at a high rate without severely inhibiting growth. The irradiation treatment given to the various plant materials is quick, lasting between a few seconds and a few minutes, but is sufficient to induce mutation. By using this method, we already put 26 new cultivars on the market in Japan, the United States, Canada, and the European Union since 2001. For example, rose induced mutations are utilized in vegetative propagated crops to shorten the time necessary to breed for desirable characters (Figure 1). In the present article, we introduce the development and practical applications of ion-beam breeding, effects of Linear Energy Transfer (LET) on lethality and mutagenesis, and future perspectives on biological applications of heavy-ion beams.

AMAP: A pipeline for whole-genome mutation detection in Arabidopsis thaliana

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.