Lixia Yu

Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

Purpose: To determine the effects of carbon ion beams with five different linear energy transfer (LET) values on adventitious shoots from in vitro leaf explants of Saintpaulia ionahta Mauve cultivar with regard to tissue increase, shoots differentiation and morphology changes in the shoots. Materials and methods: In vitro leaf explant samples were irradiated with carbon ion beams with LET values in the range of 31∼151 keV/µm or 8 MeV of X-rays (LET = 0.2 keV/µm) at different doses. Fresh weight increase, surviving fraction and percentage of the explants with regenerated malformed shoots in all the irradiated leaf explants were statistically analysed. Results: The fresh weight increase (FWI) and surviving fraction (SF) decreased dramatically with increasing LET at the same doses. In addition, malformed shoots, including curliness, carnification, nicks and chlorophyll deficiency, occurred in both carbon ion beam and X-ray irradiations. The induction frequency with the former, however, was far more than that with the X-rays. Conclusions: This work demonstrated the LET dependence of the relative biological effectiveness (RBE) of tissue culture of Saintpaulia ionahta according to 50% FWI and 50% SF. After irradiating leaf explants with 5 Gy of a 221 MeV carbon ion beam having a LET value of 96 keV/µm throughout the sample, a chlorophyll-deficient (CD) mutant, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, and plantlets with other malformations were obtained.

Mutagenic effects of carbon-ion irradiation on dry Arabidopsis thaliana seeds

To investigate the mutagenic effects of carbon ions on Arabidopsis thaliana (ecotype Columbia) and to isolate useful genes in plant development, dry seeds were exposed to 43MeV/u carbon ions at doses of 0, 100, 200, 300, 400, 500 and 600Gy. The survival rate, primary root length, and hypocotyl length of M1 plants were analyzed, and 200Gy was selected as the dose for the large-scale experiment. A total of 1363 lines of plants from 28,062 M2 populations displayed alterations in the leaf, stem, flower, or life cycle, with abnormal leaves and a premature life cycle as the main phenotypic variations. The mutated gene loci of five stable and inheritable mutations were roughly mapped on chromosomes. Novel mutants were obtained, although some of the mutants were similar to mutants induced by ethylmethane sulfonate (EMS) according to previous studies. This study provides a large body of specific information describing A. thaliana mutation phenotypes that were induced by carbon-ion irradiation. These results suggest that carbon-ion beams are as useful and effective as other mutagens for mutant breeding in plants, and that they will allow mutant breeding that is more diversified.

Identification of Substitutions and Small Insertion-Deletions Induced by Carbon-Ion Beam Irradiation in Arabidopsis thaliana

Heavy-ion beam irradiation is one of the principal methods used to create mutants in plants. Research on mutagenic effects and molecular mechanisms of radiation is an important subject that is multi-disciplinary. Here, we re-sequenced 11 mutagenesis progeny (M3) Arabidopsis thaliana lines derived from carbon-ion beam (CIB) irradiation, and subsequently focused on substitutions and small insertion-deletion (INDELs). We found that CIB induced more substitutions (320) than INDELs (124). Meanwhile, the single base INDELs were more prevalent than those in large size (≥2 bp). In details, the detected substitutions showed an obvious bias of C > T transitions, by activating the formation of covalent linkages between neighboring pyrimidine residues in the DNA sequence. An A and T bias was observed among the single base INDELs, in which most of these were induced by replication slippage at either the homopolymer or polynucleotide repeat regions. The mutation rate of 200-Gy CIB irradiation was estimated as 3.37 × 10−7 per site. Different from previous researches which mainly focused on the phenotype, chromosome aberration, genetic polymorphism, or sequencing analysis of specific genes only, our study revealed genome-wide molecular profile and rate of mutations induced by CIB irradiation. We hope our data could provide valuable clues for explaining the potential mechanism of plant mutation breeding by CIB irradiation.

Strategies for identification of mutations induced by carbon-ion beam irradiation in Arabidopsis thaliana by whole genome re-sequencing

Heavy-ion beam irradiation is a powerful physical mutagen that has been used to create numerous mutant materials in plants. These materials are an essential resource for functional genomics research in the post-genome era. The advent of Next-Generation Sequencing (NGS) technology has promoted the study of functional genomics and molecular breeding. A wealth of information can be gathered from whole genome re-sequencing; however, understanding the molecular mutation profile at genome wide, as well as identifying causal genes for a given phenotype are big challenging issues for researchers. The huge outputs created by NGS make it difficult to capture key information. It is worthy to explore an effective and efficient data-sieving strategy for mutation scanning at whole genome scale. Re-sequencing data from one laboratory wild type (Columbia) and eleven M3Arabidopsis thaliana lines derived from carbon-ion beam irradiation were used in present study. Both the number and different combinations of samples used for analysis affected the sieving results. The result indicated that using six samples was sufficient to filter out the shared mutation (background interference) sites as well as to identify the true mutation sites in the whole genome. The final number of candidate mutation sites could be further narrowed down by combining traditional rough map-based cloning. Our results demonstrated the feasibility of a parallel sequencing analysis as an efficient tool for the identification of mutations induced by carbon-ion beam irradiation. For the first time, we presented different analysis strategies for handling massive parallel sequencing data sets to detect the mutations induced by carbon-ion beam irradiation in Arabidopsis thaliana with low false-positive rate, as well as to identify the causative nucleotide changes responsible for a mutant phenotype.

Research of photosynthesis and genomewide resequencing on a yellow-leaf Lotus japonicus mutant induced by carbon ion beam irradiation

Carbon ion beam is an advanced physical mutagen that is used for mutation breeding and functional genomics research. Using carbon ion beams, a stable Lotus japonicus mutant C16 which showed yellow leaves and reduced height compared with the laboratory wild type was obtained in our previous study. Yellow‐leaf mutation is a desirable characteristic for studying photosynthesis. The photosynthetic‐related physiological indexes, including the content of chlorophylls and the ratio of variable fluorescence (Fᵥ) over the maximum fluorescence value (Fₘ) (maximal photochemical efficiency of photosystem II in the dark) of leaves between laboratory wild type and C16, were investigated. C16 showed lower amount of chlorophylls than that of laboratory wild type, as well as a relatively photosynthetic capacity. Meanwhile, to comprehend the molecular mechanism underlying the mutant, genomewide resequencing was performed to identify the mutational site responsible for the yellow‐leaf phenotype causing the weaker photosynthetic capacity. Ninety‐five single‐base substitutions and 17 small insertions and deletions were found in C16 mutant. Among those, by association analysis of functional annotation and mutant phenotype, T>G SBS in chromosome 5 located in Lj5g3v1669360.1 which encodes arabinogalactan protein was supposed as candidate mutation sites. Quantitative real‐time polymerase chain reaction analysis indicated that the expression level of Lj5g3v1669360.1 in C16 was significantly lower than that of laboratory wild type. Decreased expression of the Lj5g3v1669360.1 may be responsible for the smaller amount of chlorophylls causing yellow leaves, the weaker photosynthetic capacity of C16, and resulting in slow growth.