Mapping Of QTLs For Accumulation Of Cadmium In Rice Grains And Breeding Application Of Related Genes

Rice is an important food crop,and more than half of the world’s population uses rice as the main food;compared with other food crops,rice can absorb and accumulate more cadmium,and reducing the cadmium content in rice is an effective measure to ensure food safety.The rice fields polluted by cadmium occupy a large proportion in different degree in South China,and the events of rice polluted by cadmium occur frequently.Breeding rice varieties with low cadmium accumulation is an important goal of rice breeding.Therefore,exploring low cadmium accumulation rice materials and related genes are the basis of breeding rice varieties with low cadmium accumulation.In this experiment,the RIL lines of R3551 and R498,which had the same growth period and plant height,were used as materials to analyze the main QTL locus controlling the accumulation of cadmium in rice grain and to locate it precisely.On the other hand,three cadmium accumulation related genes,OsLCT,LCD、OsNRAMP5,were knocked out in different background materials,and homozygous mutants with double knock and multiple knock were screened to explore the genetic effect of the mutants on cadmium accumulation and the impact of agronomic traits.The main results are as follows:1We investigated the roots,shoots and mature seeds Cd concentration among 410 RIL population materials constructed by R498 and R3551,A totle of 5 QTLs associated cadmium accumulation in the RILs populations combined with the high-density genetic linkage bin map using composite interval mapping.A QTL,qCdR6 relating to cadmium uptake by seedling roots was found on chromosome6;Two QTLs,qCdL6 and qCdL7 relating to cadmium transfer in shoot of seedling were found on chromosome6 and 7;Two QTLs qCdGl and qCdG7 related to the accumulation of cadmium in grains on chromosomes 1 and 7.A major-effect QTL,qGCd7 is located between 8.4-8.9 Mb on chromosome 7 and has a genetic effect of 22.67%.In the positioning interval,there is a gene related to cadmium absorption OsNRANP5.There is a SNP difference between the parent material in R498 and R3551 in the tenth exon of the coding region and 4 SNP differences in the 2K promoter region,which does not affect translation.2.Three genes related to cadmium accumulation,OsHMA3,OsNRAMP1 and OsNRAMP5,have been reported near the location of qCdG7 Near-isogenic lines N4019,N4022 with two different genotype combinations of R498 background were screened by BC4F3:OsHMA3R498OsNRAMP5R3551OsNRAMPIR3551(4019),OsHMA3R3551OsNRAMP5R498OsNRAMP18498(4022).Under the condition of hydroponics at seedling stage,the content of Cd in the shoot of 4019 and 4022 was significantly lower than R498,and the content of Cd in the root of 4019 was also significantly lower than R498,while that of 4022 was significantly higher than R498.In the field experiment,there was no significant difference between the cadmium content of 4019 and R498,which was significantly lower than that of R3551.The expression level of OsNrampl and OsNramp5 in 4019 was higher than that in R498,but there was no significant difference in the level of OsHMA3.The expression level of OsNrampl and OsHMA3 in 4022 was significantly lower than R498,but the level of OsNramp5 was no significant3.We use CRISPR/Cas9 system to knock out OsNRAMP5,OsLCT and LCD and obtained homozygous lines of tri-and bi-processes(osnramp5/oslct).In the fields contaminated with Cd,the cadmium content in the brown rice of mutants KO-1 and KO-2 decreased by 87.5%and 83.7%,respectively,and the reduction in stalks and leaves was 91.6%,the difference was very significant;There was no difference in plant morphology,and there was no significant difference in plant height,tiller number,seed setting rate,thousand-grain weight,grain length,and grain width,and its yield and main agronomic traits were not affected.In the hydroponic experiment at the seedling stage,the cadmium content decreased most significantly at 2.1μM,KO-1 decreased by 75.6%,KO-2 decreased by 58.6%,the Mn content of the knockout lines showed a downward trend,and the Zn content did not change much.