Characterization And Target-select Identification Of Genomic Variations Induced By Nuclear Radiations In Rice

Nuclear radiation as a breeding means has played a great role in germplasm innovation and crop varieties development. However, knowledge of induced variations at molecular level, particularly at genome level, has been scare, which has restricted the advance of radiation breeding. To reveal the mutagenic effect and establish efficient mutation breeding systems, the genomic variations induced byy rays and high energy ion beam radiations were systematically investigated in the present stdy, using genomic techniques (e.g. genome resequencing) and molecular means in rice, and two new techniques, i.e. next generation sequencing (NGS) and high resolution melting (HRM), were explored for screening deletion mutations. The main results are summarized as following:1. The characteristics and frequency of inheritable mutations induced by γ rays were revealed at the whole genome level with single nucleotide resolution. Not only single base substitutions (SBSs) but also insertions and deletions (Indels) and structure variations (SVs) were induced by y rays. Mutations were detected at the average frequency of 7.5-9.8×10-6 per generation per site in six sequenced M2 rice plants. SBSs are about 4 times more frequent than Indels; structural and copy number variations, though less frequent, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots.2. The biological and mutational effect of three high energy ion beams (C, Ne and Ar) were comparatively investigated in rice, and the suitable doses for mutation induction and types of mutation induced were determined. Ar ions radiation had greater effect than C and Ne ions (the latter two were similar) on survival rate and seed-set of Mi rice plants. The shoulder dose, which is often regarded as thesuitable dose for efficient mutant development, was determined for the three ion beams; It was observed that M2 panicles of a common M1 plant could carry different mutations; SBSs and Indels mutations were induced by ion beams and were scattered across 12 rice chromosomes.3. The effectiveness of NGS- and HRM-based methods (Seq-TILLING and HRM-TILLING, respectively) for high thoughput mutation screening and influencing factors were investigated. When Seq-TILLING was applied for indel mutation screening, false positive mutations were reported, but they could be filtered out by further bioinformatics analysis; sample pooling at 192×is suitable; In HRM- TILLING analysis, very low false positive or negative rates were observed for short indels when samples were pooled at 4 times, however, it became less effective for fragments with long indel mutations.The findings of the present study on y rays mutagenesis provided us scientific guidance for the design and implementation of mutation breeding programmes, e.g. the similar mutation rates of plants irradiated with different doses of y rays suggest that a relatively lower dose than commonly used could be applied, the existence of thousands of induced mutations in each M2 plant suggested that selected mutants should be subjected to further breeding by backcrossing to clear the deleterious mutations; and single seed selection, instead of bulk selection, should be applied in mutant line development because background mutations may still be segregating in M3 and advanced generations. The determination of suitable dose and types of mutations induced, as well as different mutations discovered in different panicle rows of a common M1 plant, laid a solid foundation for establishing mutation breeding systems for the three ion beam. The establishment of Seq-and HRM-TILLING platform for high throughput screening of target-select mutations is expected to foster the use of mutation breeding.