| Due to its unique physical and biological advantages, heavy ion beam, with high mutation rate and wide mutation spectrum, has been widely used in plant breeding as a novel and efficient physical mutagen. At present, because of the unclear genetic backgrounds of many crops, the research about the mechanism of radiation breeding is relatively limited. In this study, dry Arabidopsis thaliana seeds(ecotype: Columbia) irradiated by carbon ion beams were used for mutant screening. The quantitative statistical analysis of mutation rate and mutation spectrum in M2 population were performed to assess the mutagenic effects of carbon ions at the phenotypic level. Meanwhile, the phenotypic, physiological and biochemical characteristic of stable mutant #197 obtained by using carbon ion radiation mutation technique, as well as the resistance to abiotic stresses, were investigated. In addition, based on the whole-genome resequencing technology, the variations of mutant #197 at DNA level had been analyzed and identified. To explore the mutant genes that controlled the mutation phenotypes, map-based cloning technology was undertaken to narrow the search interval. The results, obtained from phenomics and genomics, will enrich the understanding of the molecular mechanism and biological effectiveness of carbon ion irradiations on Arabidopsis thaliana, thus may provide the theoretical foundation for heavy ion radiation mutagenesis on plants.Dry Arabidopsis thaliana seeds were exposed to carbon ion beams with energy of 43.3Me V/u and 10.3 Me V/u(The average LET value within sample as water equivalent material was calculated to be 50 ke V/μm and 200 ke V/μm, respectively) at the room temperature and atmospheric conditions. The biological effects of carbon ion irradiation on the M1 seeds of Arabidopsis thaliana were investigated firstly. Except the plant height, all the indicators including survival rate, root length, hypocotyls length and number of seeds per silique were decreased with the increasing of dose at the same LET condition. Meanwhile, the extents of the decline of survival rate, root length, hypocotyls length and number of seeds per silique were reinforced as the increasing of LET. In order to achieve the higher mutation rate, stable and hereditable mutants, the optimized parameters of carbon ion beams used for the large-scale mutation screening of dry Arabidopsis thaliana seeds were 200 Gy at LET value of 50 ke V/μm.In total, 1363 plant lines that displayed macroscopic variations were isolated as the putative mutants from the M2 population, and they were divided into four groups according to their phenotypes: 1) Mutations in the leaf morphology, 2) Mutations in the stems, 3) Mutations in the flowers, 4) Mutations in the life cycle. Each category was subdivided into several sub-groups. The total mutation rate was 4.77%, the occurrence of leaf mutants and life cycle mutants were 2.33% and 1.57%, respectively, while that of stem and flower mutants were only 0.27% and 0.13%. In addition, the statistical analysis of pleiotropic phenotypes was performed as well: in the 1363 mutant lines, 64% displayed one conspicuous phenotype, 13.3% displayed two conspicuous phenotypes, and 13.1% showed at least three mutant traits.The phenotypes of the stable and hereditable mutant #197 which was induced by the carbon ion irradiation were studied intensively. All indicators including the root length, hypocotyls length, fresh weight and plant height of #197 were lower than wild type(WT) plants during the vegetative growth stage. According to the statistical results of phenomics, the total leaf area of #197 was much smaller than WT, as well as the leaf fluorescence intensity of #197 was weaker than WT. As for the morphology, the first three pieces of true leaves of #197 were larger than those of WT. Meanwhile, #197 also displayed thin, transparent and pale green leaves during the early growth, and then became concave and convex. The internal cytological structure of #197 leaves had varied correspondingly. Compared with WT, the #197 showed lower activities in PSII, due to its decreased chlorophyll fluorescence parameters, such as Fv/Fm, ΦPSⅡ and q P. In addition, #197 performed later flowering time, weaker pollen vitality as well as stunted siliques during the reproductive growth stage.Based on the whole-genome resequencing technology, the variations of #197 at DNA level had been analyzed and identified. According to the statistical analysis, 590 million paired-end reads which covered 49 folds of the reference genome were generated. A total of 4642 SNP and 3071 In Del, 79 CNV and 288 SV were identified in #197. Interestingly, SNP and In Del tended to occur in the pericentromeric regions in each chromosome as clusters, especially in chromosome 1. The ratio between the A-G, T-C transition and A-C, A-T, G-C, G-T transversions was 0.996:1. In addition, the GO annotation and classification for the functional genes which showed variations were also performed in this study. The results showed that 47% of the mutational genes were related to the biological process; and 27% of the mutational genes were involved in the cellular component; 26% of the mutational genes performed molecular function. By using the map-based cloning technology, the mutational genes which controlled the phenotypes of #197 were positioned in the ranges of T23K8 to F4N2(1832kb) on chromosomes 1 and F6I18 to F8B4(579.773kb) on chromosomes 4. Based on the association analysis of whole-genome resequencing and map-based cloning, there were a total of 32 candidate genes located in the aboved regions.The sensitivities of #197 and WT to Na Cl and drought stress were compared in this study as well. Salt stress significantly delayed the seed germination of WT and #197, but did not restrain the final germination rates of Arabidopsis thaliana seeds. During the seedling stage, when stressed by Na Cl, the green cotyledon rate of #197 was much higher than those of WT, which indicate that #197 was less sensitive to Na Cl than WT. The expression level of P5CS1 of #197 under salt stress was significantly higher than that of WT. Under normal conditions, there was no difference in water content between WT and #197. When stressed by drought, the rates of water loss in #197 under drought stress were higher than that of WT. Meanwhile, the proline content of #197 was lower than that of WT, as well as the expression levels of certain key marker genes which could response to drought stress in #197 were much lower than those of WT. However, the phenotypic discrepancies between # 197 and WT were not significant.In summary, 1) Carbon ion beam irradiation showed significant biological effects on the M1 seeds of Arabidopsis thaliana. The seeds irradiated by carbon ion beams with the LET value of 50 ke V/μm and the dose of 200 Gy were chosen to be performed in the large scale mutation screening. 2) The total mutation rate of carbon ions in M2 populations was 4.77%, while the mutation spectrum ranged from variations in leaf, stem and flower to mutations in life cycle. In addition, the mutations of Arabidopsis thaliana induced by carbon ion beam irradiation had obvious pleiotropic phenotypes effects. 3) Compared with WT, #197 manifested conspicuous differences in morphology at both vegetative and reproductive growth stages, which showed a delayed and weak trend in growth and development. 4) Various kinds of mutations at DNA level, such as inversion, translocation, insert, delete and SNP, occurred in #197. 5) The mutational genes which controlled the phenotypes of #197 had been mapped on chromosomes 1 and chromosomes 4. 6) The #197 was less sensitive to Na Cl than WT at seedling stage. Under drought stress, the physiological indicators showed remarkable discrepancies between #197 and WT, as well as the expression levels of stress response genes. While there were no significant differences in phenotypes between #197 and WT. |
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