Study On Mutagenic Effects Of Carbon Ion Beam Radiation On Rice

Rice（Oryza sativa L.）is not only an important food crop but also an important model species for monocotyledonous plant biology research.With the increase in world population and climate change,food security issues are becoming increasingly prominent.Therefore,the demand for rice breeding is constantly increasing worldwide.Traditional breeding methods are difficult to produce new genetic resources,but mutation not only directly provides rich rice germplasm resources for breeding,but can also be used in combination with hybrid breeding and molecular breeding for breeding.At the same time,mutation also provides materials for functional genomics research.Heavy ion beam（HIB）is an efficient physical mutagen with unique physical and biological characteristics.It is increasingly used in plant mutagenesis breeding and mutation population construction.However,previous studies of HIB mutagenesis effects were mostly limited to certain generations,and there are few reports of continuous tracking of HIB radiation-induced mutagenic effects from the M₁ to the high generation.Systematically revealing the multi-generation and multi-dimensional mutagenic effects of HIB radiation on rice can improve the basic theory of HIB mutagenesis and contribute to its more efficient application.In this study,Kitaake,a model rice variety,was used as the research object.Samples were treated with carbon ion beams（CIB）of different absorption doses,and the biological and mutagenic effects of the M₁-M₄ genetic generations after irradiation were systematically studied.The main results are as follows:1.When the radiation dose exceeds 125 Gy,it is considered a high dose for Kitaake.Ten doses of CIB（25-300 Gy）were used to irradiate Kitaake dry seeds,and the biological effects induced by different doses of radiation during the entire life cycle of the M₁ generation were studied.It was found that CIB have a clear dose-effect relationship,and radiation doses higher than 125 Gy can cause significant growth damage to rice.Specifically,at this dose,the root length and shoot length of seedlings were only 24.8%and 39.3%of the control group,respectively,and the survival rate was only 34.2%of the control group.Moreover,the chlorophyll content during the tillering stage was only about 80%of the control group,and the chlorophyll fluorescence parameters were also significantly affected.At the maturity stage,the plant height was lower than that of the control group by 11%,the tillering and panicle numbers were doubled that of the control group,and the grain setting rate was less than 25%.2.CIB mainly induces SBSs and<10 bp In Dels,and the largest number of mutations are induced at 100 Gy.Based on the biological effects of rice in the M₁ generation,six doses of CIB treatment groups（25-150 Gy）were selected to reveal their genome variation characteristics induced in M₂ seedlings.Single base substitution（SBSs）was the most abundant type induced by CIB（approximately 72%）,followed by deletions（approximately 23%）,and insertions were the least abundant（approximately 5%）.The largest number of mutations were induced at 100 Gy,with an average of 105.23±2.83per plant,and a mutation rate of 2.67×10^-7/bp.<10 bp deletions accounted for 93.19%of all mutations,and<10 bp insertions accounted for more than 99%.In the M₂generation,the ratio of homozygous mutations to heterozygous mutations induced by irradiation was 7:3.CIB-induced transitions（Ts）were more abundant than transversions（Tv）,and the ratio of Ts to Tv induced at various doses ranged from 1.17to 1.50,with an average of 1.28.3.Different panicles from the same M₁plant may originate from different progenitor cell.The common mutations from different panicles of the same M₁ plant were counted,and it was found that the common mutation rate among the three panicles from the same M₁ plant was very low,at around 7.5%,and the common mutation rate between two panicles from the same M₁ plant was only 20%.The rate of unique mutations in individual panicles was very high,averaging 66.9%.Therefore,there are still a large number of unique mutations in different panicles from the same M₁ plant,indicating that different panicles may originate from different progenitor cell during development.4.There are differences in the variation characteristics of different genetic generations.Comparing the variation characteristics of M₂ and M₄ generations,it was found that the proportion of SBSs and insertions increased,while the proportion of deletions decreased in the M₄ generation.The proportion of homozygous mutations also increased,with a ratio of 5:2.The Ts/Tv value was also higher in the M₄ generation.5.High-generation stably mutants can be used to preliminarily predict candidate mutated genes using WGS technology.Using whole-genome sequencing（WGS）technology to reveal the variation sites of eleven phenotype-stable rice M₄ mutants,candidate genes controlling the phenotype variation were predicted through annotation of the mutant sites.Overall,this study revealed the mutagenic effects of CIB from multiple dimensions using multiple genetic generations.It improved the research on mutagenic effects of CIB and provided theoretical support for its more efficient application in mutagenesis breeding.In addition,screening a large number of Kitaake mutants enriched genetic resources and provided basic materials for genetic analysis,functional genomics,and breeding research.Additionally,this study provides scientific evidence for setting an appropriate dose of HIB mutagen.