Studies On Mutations Induced By Physical Radiations In Rice Nuclear Genomes,Cytoplasmic Genomes,and Epigenomes

Physical radiations are essential means for inheritable mutation induction and mutant population construction in plants.Representative nuclear radiations,like gamma(?)ray,ion beam,etc,have been widely used for germplasm innovation and crop breeding,which thus made radiation breeding a branch of crop breeding.In recent years,some studies have been carried out exploring the inheritable mutations induced by nuclear radiations on the whole-genome scale and at the single-nucleotide resolution.However,such kind of studies were limited to Arabidopsis,rice,etc,with an inadequate number.Meanwhile,the plant materials and analytical methods used in these studies were not the same,and the conclusions obtained from different studies were not in a good consistence.In addition,cytoplasmic genomes and epigenomes also play an important role in trait development.Nevertheless,up to now,there have been no reports on cytoplasmic and epigenomic variations induced by radiations in plants.Therefore,more and deeper studies are necessary to reveal the characteristics of radiation-induced genomic variations and the molecular mechanisms of mutation formation,so as to provide genomics support for promoting the theoretical progress and method innovation in plant mutation breeding.High energy ions are considered to be a new kind of physical mutagen,which are different from gamma rays.Different ions have different linear energy transfers(LETs),and the efficiency of mutation induction could be significantly different.In rice,the mutagenic effect of carbon(C)ions has been studied while that of argon(Ar)and neon(Ne)ion beams on rice has not been elucidated.Therefore,the present study firstly elaborated the physiological and mutagenic effects of Ar,C and Ne ion beams on rice.Then the mutations and their characteristics induced by the three ion beams in nuclear and cytoplasmic genomes were identified and investigated by whole genome re-sequencing,and compared with the mutations and their characteristics induced by?rays in cytoplasmic genomes.Besides,the epigenomic variations in two?ray induced rice mutants and their progenitor were studied by whole genome bisulfite sequencing.The main findings are summarized as follows:Ar,C,and Ne ion beams showed damage effects on M₁plants,e.g.,the half lethal dose of Ar ion beams was?100Gy while that of C or Ne ion beams was required to be?200Gy or higher;moreover,Ar,C,and Ne ion beams could result in high frequencies(up to?30%on M₁plant basis)of morphological mutations in M₂plants.All the three ion beams could significantly reduce the survival rate of rice seedlings at higher dose of treatment and severely damage seedling growth of M₁plants,with significant variation among ion beams and doses,as well as genotypes.In M₂populations,high frequencies of chlorophyll deficient seedlings and male sterile plants were observed.Ar,C and Ne ion beams could all effectively induce single base substitutions(SBSs)and insertion/deletions(In Dels)with similarly high mutation frequencies observed in rice M₄plants when proper doses are applied(e.g.,Ar ion beams at?100Gy).There were 236-453 mutations in each of the six rice M₄plants derived from the three ion beam radiations,including SBSs and In Dels,with a?4-8 times greater number of SBSs than In Dels.SBS and In Del mutations were distributed not evenly across all 12 chromosomes with a few mutation peaks.And only a small number of mutations(0-6)were exonic ones that might affect gene function.Ar,C and Ne ion beams could all induce structural variations(SVs)with different formation mechanisms.The number of identified and verified inheritable SVs induced by all the three ion beams in each of the six rice M₄plants ranged from 1to 4,including deletion,duplication,intra-chromosomal and inter-chromosomal translocation.All the SVs were distributed across the whole genome with specific characteristics around SV breakpoints,based on which the SVs were deduced to be formed through the expansion or contraction of tandem repeat,insertion of transposable elements,and non-homologous recombination.Gamma ray and Ar,C,Ne ion beams could induce SBSs and In Dels in both chloroplast(cp)and mitochondrial(mt)genomes,and mutation frequencies might differ greatly from plant to plant.The majority of identified mutations were background ones in the six?ray irradiated M₂and six ion beam irradiated M₄plants and most of them were heterogeneous.Though the induced mutations were distributed in different genomic regions,no one was annotated to cause nonsynonymous mutations or frameshifts among the M₂plants,whereas four SBSs,two each in cp and mt genomes of the M₄plants,were nonsynonymous mutations.Genome-wide epimutations were identified in two?ray induced rice mutants.Huangyu B(HYB)is a xantha leaf mutant derived from?ray irradiated Longtefu B(LTB),while Cuiyu B(CYB)is a leaf color reverse mutant derived from?ray irradiated HYB.The study showed that the epigenomes of the three rice lines were somewhat different.The two mutant rice lines HYB and CYB were diverged from the progenitor line LTB with slightly more hyper-methylated differentially methylated positions(DMPs)and regions(DMRs)than hypo-methylated ones,and CYB was more different from LTB than from HYB.More DNA methylations were present in the CG context than CHG and CHH,and enriched in intergenic regions,promoters and transposable elements(TEs)in rice.A total of 80 DMPs were probably involved in the rice leaf color changes.In summary,a number of innovative findings have been obtained in the present study on radiation induced mutations in rice.Firstly,the biological and mutagenic effects of Ar and Ne ion beams on rice were revealed,including the type,frequency and characteristics of mutations induced by the two ion beams in the nuclear genomes,and the similarities and differences when compared with those of C ion beams.Secondly,the main characteristics of structural variations(SVs)induced by the three ion beam radiations in the rice genomes were uncovered,and the molecular mechanisms of SV formation were relatively fully understood,which is a pioneer one of such kind of research in plants.Thirdly,the cytoplasmic mutations induced by?rays and high-energy ion beams in rice were discovered,which is also the first research on mutagenic effects on cytoplasmic genomes.Fourthly,the characteristics of epigenomic mutations induced by?rays were revealed on the whole-genome scale and at the single-nucleotide resolution,which sets a precedent for the studies of epigenomic mutations induced by radiations in plants.These findings are of great significance for the construction of scientific theories,technologies and methodologies of radiation mutation breeding,especially for the genomics theory systems of radiation breeding promoting a more effective and efficient utilization of?ray and ion beam irradiation for the development and application of rice and other crop mutants.