Biological Effect Analysis Of Space Mutagensis And Heavy Ion Radiation And Efficient Targeted Screening Of Mutants In Rice

The innovation of rice germplasm resources is the basis of the breeding of new rice varieties,and it is of great significance to use various types of mutagenic methods combining with modern biology to innovate the excellent germplasm of rice.Spatial mutagenesis is a combination of China’s space biology and rice genetics and breeding.It has played a prominent role in rice germplasm innovation and variety breeding.Heavy ion radiation is an effective simulation method for space radiation and is of great value for broadening space mutation breeding applications in rice.At present,a large number of studies have been carried out on the spatial mutation of rice,but there is still a lack of report on the comparision of the biological effects between heavy ion irradiation and space mutation on rice.In addition,for a large number of individuals obtained after mutagenesis,how to efficiently and accurately identify different types of mutants,and then build a mutant library to serve breeding practice and theoretical research,is essential for improving rice mutation breeding efficiency.In this study,space mutagenesis and heavy ion mutagenesis were used to mutagenize dry seeds of Huahang 31,and mutagenic effects of phenotype and cytology were analyzed.For the mutagenized offspring,this study investigated the genotype-directed screening techniques,targeted phenotypic and genotypic directional screening for amylose content and grain type,and obtained a series of candidate mutants.The main findings are as follows:1.Compared with spatial mutagenesis and heavy ion mutagenesis（80Gy）,space mutagenesis had a certain stimulatory effect on the germination of Huahang 31 seeds,but the growth of shoots was inhibited.There is no significant difference of the main agronomic traits of mature stage compared with those of the control.Heavy ion mutagenesis has significantly more physiological damage to Huahang 31 than spatial mutagenesis,and its significant reduction in seed setting rate has seriously affected the yield per plant.2.The mutagenic effects of different doses of heavy ion mutagenesis（5Gy,10Gy,20Gy,40Gy,80Gy,160Gy）on Huahang 31 were obviously different and had certain rules.The seed germination potential and shoot length showed typical saddle-type dose-response curves with increasing doses.Physiological injury showed a trend of increasing first and then decreasing again with increasing dose.The physiological injury was the lowest at 40 Gy.Low-dose heavy ion mutagenesis had no significant effect on the mutagenic contemporary agronomic traits,and high-dose heavy ion（80 Gy and above）mutagenesis had a significantly lower rate of seed setting.3.Heavy ion mutagenesis led to mitosis and chromosomal abnormalities in rice root tip cells.With the increase of mutagenic dose,the mitotic index of rice root tip decreased and the micronucleus fraction increased.During mitosis,abnormalities such as chromosome fragments,chromosome bridges,backward chromosomes,and inhomogeneous chromosome divisions can be generated.The chromosome aberration rate increases with increasing dose of mutagenesis.4.The TILLING-HRM system was optimized to make it suitable for the mutated genotype-directed screening of this study.The annealing temperature of the nested PCR primers used for HRM detection was determined,and by comparing the HRM detection effects of the DNA mixing pools of different magnifications,It was decided to use 4 times as the optimal magnification for mutation screening.Four pairs of nested PCR primers for scanning for Wx gene mutations were obtained by design and screening,and there were 1184spatial mutagenesis SP₂ DNA mixing pools and 1212 heavy ion mutagenesis M₂ DNA mixing pools were screened by TILLING-HRM.Finally,a heterozygous mutant obtained by heavy ion mutagenesis was selected,and the mutation type was a missense mutation of C?T.5.The grain type performance of 4908 space mutagenesis and heavy ion mutagenesis second generation rice plants were investigated.A total of 62 candidate mutants were screened from space mutagenesis with a total mutation rate of 3.10%.A total of 142 candidate mutants were screened from heavy ion mutagenesis,with a total variation rate was 4.88%.The mutagenic material from space mutagenesis is mostly shortened in grain length,widened in grain width and smaller in the ratio of length to width,while the mutagenic material from heavy ion mutagenesis is mostly longer,narrower,and the ratio of length to width is larger.6.The content of amylose in 4881 second generation rice plants induced by space mutagenesis and heavy ion mutagenesis was screened by near-infrared reflectance spectroscopy.A total of 39 candidate mutants were screened by space mutagenesis,and the mutation rate was 1.63%.A total of 38 candidate mutants were screened by heavy ion mutagenesis,with a mutation rate of 1.53%.The number of mutants with high amylose content in space-induced mutagenesis was twice that of mutants with low amylose content,but the number of mutants with high amylose content induced by ion mutagenesis was not different from the number of mutants with low amylose content.In summary,this study compared the mutagenic effects of space mutagenesis and heavy ion mutagenesis（include 80Gy heavy ion mutagenesis and different doses of heavy ion mutagenesis）on Huahang 31;The 40Gy heavy ion mutagenesis has low physiological damage and high mutagenic efficiency,which is similar to the spatial mutagenesis of this time.Combining TILLING techniques with HRM detection methods for rice space mutation and heavy ion radiation mutation screening was carried out to achieve mass screening of target gene mutations based on"sample mixed pool".Multiple rice quality mutants with grain type and amylose content were screened.The research results are helpful to understand the difference of biological effects of space mutagenesis and heavy ion radiation,and have certain theoretical value;the established genotypic orientation screening technology flow has a strong guiding significance for guiding efficient selection of mutagenized offspring.