Germplasm Creation And Study On The Mechanism Of Petal Senescence Delayed Mutant Of Lotus Japonicus Irradiated By Mid-energy Carbon Ion Beams

Mid-energy heavy ion beam mutation breeding has the beneficial characteristics of high mutation rate and wide mutation spectrum,and is widely used in plant mutation breeding.Ion beam mutagenesis breeding of leguminous plants has been carried out and achieved fruitful results,but the study of its mutagenesis mechanism is still limited.In this study,the model plant of legume,Lotus japonicus?L.japonicus,ecotype:MG-20?,was used as the research material.Dry seed of L.japonicus were irradiated by carbon ion beams,and the population of M₂ generation was selected for mutant screening.The mutation rate and mutation spectrum were analyzed quantitatively,and the genetic polymorphism of irradiated progeny was represented by ISSR technology.The Petal senescence delayed?PSD?mutant was obtained from the M₂ population,and the variation loci and differentially expressed genes of PSD were searched by next generation sequencing?NGS?and transcriptome sequencing analysis.The gene regulation network of petal senescence process in L.japonicus was explored by parsing the transcriptome data in-depth.Dry seeds of L.japonicus were irradiated by 80MeV/u carbon ion beam provided by Heavy Ion Research Facility in Lanzhou?HIRFL?,the radiation dosages were 0,100,200,300,400,500 and 600 Gy,respectively.The germination rate,survival rate and root length were analyzed statistically.It was found that there were no relations between germination rates and absorb dosage,but the survival rate and root length decreased with increasing doses.Finally,400 Gy was selected as lethal dose,50%?LD50?.1200 M₁ seeds were irradiated by carbon ion beams with this dose,and 127individuals with phenotypic variation were screened from 2,472 offspring.The mutation types of the mutant population included leaf mutations,stem mutations,floral mutations,and fruit mutations.The total mutation rate was 5.14%.Then the inter simple sequence repeat?ISSR?molecular marker technique was used to perform the study of genomic DNA polymorphism in M₂ plants.Seven individuals with mutational phenotype and eight individuals without mutational phenotype in M₂population were analyzed by ISSR.There were 4 polymorphic bands in total.There were 2 polymorphic bands among the seven mutants,as well as two polymorphic bands among the eight M₂ plants without phenotypic variations.The polymorphic rate were 0.607%and 0.532%,respectively.No remarkable differences were detected between these two groups,and the total polymorphic rate was 0.567%.There was no report on the PSD mutant in L.japonicus obtained from the offspring population irradiated by carbon ion beams.The senescence of the mutant petals is delayed significantly,which has great research value and potential economic value.The results showed that the flowering period of a single flower in PSD was longer than that of wild-type?WT?for more than 10 days.The pod length of PSD was slightly shorter than WT and the number of seeds in one pod was slightly less than that of WT,but did not reach a significant difference.The growth cycle,plant morphology,leaf shape and flower pattern of PSD mutant were no significantly different from those of WT.The results of trypan blue staining and semithin sections of petals showed that after pollination,the survival time of petal cells in PSD was longer than that of WT.After wilting,the structure of petal cells in PSD was relatively intact,the area of trypan blue staining was less than that of WT,and the cell structure was more complete than WT.By re-sequencing and transcriptome association analysis,it was estimated that CUFF.19232 is a candidate gene that causes prolonged flowering of the PSD.CUFF.19232 was annotated as papain-type cysteine endopeptidases?CysEPs?cotaining a C-terminal KDEL endoplasmic reticulum?ER?retention signal?KDEL CysEPs?.CUFF.19232 shares the 82%similarity with AT5G50260?KDEL-CysEPs?in Arabidopisis.The specific function of this gene needs further study.Petal senescence is one of the important processes of plant growth and development.The underlying mechanism of petal senescence of in L.japonicus is still obscure.In this study,the wild-type?WT?of L.japonicus was used as the main research material to explore the genes involved in the senescence of petals,and PSD mutants was used as verification.Through the analysis of differential expressed genes in WT and PSD,four key senescence-associated genes?SAGs?and 22ubiquitination-related genes were screened to participate in petal senescence in L.japonicus.Seventy-four transcription factors?TF?family were involved in the signal transduction of petal senescence in L.japonicus,of which the AP2-ERFBP family members were the most abundant.Seven plant hormone-related genes involved in petal senescence of L.japonicus were screened.Among them,the key genes for ethylene synthesis,ACS1 and ACO1,were up-regulated and the expression of multiple ethylene-responsive TFs?RAP2-3,ERF010,ERF060 and ERF112?was up-regulated.The RT-PCR results for these genes were consistent with the transcriptome sequencing results.In vitro spraying of an ethylene biosynthetic intermediate,1-aminocyclopropane-1-carboxylic acid?ACC?,and an anti-ethylene agent,silver thiosulfate?STS?,experiments showed that ACC can accelerate the process of the petal senescence;STS can delay petal senescence effectively.Therefore,it was demonstrated that ethylene play a key role in petal senescence in L.japonicus.Through the above studies,we established a mutant library of M₂ generation of L.japonicus irradiated by mid-energy carbon ion beams,and investigated the effects of mutagenesis on the DNA polymorphism of offspring.Through the association analysis of genome and transcriptome of mutant,it can be concluded preliminarily that CUFF.19232 was an important gene responsible for the delayed senescence of PSD petals.In addition,this study explored the petal transcriptome of L.japonicus further,and investigated the genes involved in petal senescence of L.japonicus.This study demonstrated that pollination is an important event for petal senescence,and ethylene plays a crucial role in petal senescence of L.japonicus.