Mapping RSV Resistance Genes By BSA-Seq In Arabidopsis Thaliana

Rice stripe virus（RSV）,a representative species of the genus Tenuivirus,is an RNA virus transmitted by Laodelphax Striatellus,causing extremely destructive rice stripe disease.Since the 21st century,the disease has broken out in East Asia,seriously threatening rice production.At present,the most conventional control measures are the use of chemical pesticides against L.striatellus,but pesticides are expensive and also harmful to the environment.The breeding of disease-resistant varieties is considered to be a relatively economical,safe and effective way to control the disease.Conventional breeding has a long cycle and low efficiency,and the rice genome is relatively complicated.Few RSV resistance genes cloned from rice.Therefore,it is necessary to find new genetic resources for RSV resistance.Arabidopsis thaliana is a model plant in the field of plant research.Its natural population is rich in genetic variation and has significant differences in resistance to multiple plant viruses.It is a good model for identifying virus resistance genes.In this study,A.thaliana was used to excavate RSV resistance-related genes,and ecotypes with extreme resistance/susceptibility to RSV were screened as a parental cross,and the F₂ generation population was constructed for RSV resistance identification.At the same time,the genetic loci of RSV resistance were located by BSA-seq technology,and the candidate genes related to RSV resistance were screened.The main findings of this study are as follows:1.The RSV resistance of different ecotypes of A.thaliana was identified,and the highly resistant ecotype Aa-0 and susceptible ecotype Col-0 were selected as parents for crossing,heterozygous F₁ plants were self-crossed,and F₂ population was constructed.2.The RSV resistance of F₂ population was identified,the statistical data shows that the RSV resistance segregation ratio of RSV resistance and susceptibility of this population was is about 2:1,which did not meet the trait segregation ratio of classic single gene control.It is speculated that RSV resistance in Arabidopsis may be controlled by multiple gene loci.3.Using the BSA-seq technology to construct the RSV extreme resistance/sensitivity pool for sequencing.The total Raw data was 11.582 G,and the filtered Clean data was 11.564G,in which Q20≥97.55%,Q30≥93.06%,GC content was between 38.01%and 38.23%,the comparison rate between the sample and the reference genome is between 96.51%and97.54%,and the 1 X coverage is above 99.97%.Using SNP-index and In Del-index algorithms to locate the target interval on chromosomes 3 and 5.4.According to the function annotation analysis of candidate genes,12 candidate genes related to RSV resistance were screened,namely ATCPSP、REM13、ATRPSR、ATPKFP、ATSNEF、KING1、ATPSPS、ATPKSP、ATLRPK、WNK3、CYP71A26 and ATARSP.5.Purchasing candidate gene T-DNA insertion mutant seeds and carring out molecular identification of these mutants to screen out homozygous T-DNA insertion mutant strain of wnk3,pkfp,cpsp-1,cpsp-2,pksp,lrpk-1,rem13-2,arsp-1 and rpsr.RT-PCR analysis showed that the ATRPSR gene in the rpsr mutant was completely knocked out and was a loss-of-function mutant;the transcription level of the ATCPSP gene in the cpsp-1 mutant was reduced.RSV resistance identification results showed that compared with Col-0,the susceptibility of rpsr mutant plants had no significant change,while cpsp-1 mutant plants showed disease resistance.ATCPSP may be involved in the RSV resistance mechanism.In summary,the study used A.thaliana as material and used BSA-seq technology to screen out 12 genes related to RSV resistance.Through RSV resistance identification,it is speculated that that ATCPSP may be involved in the RSV resistance mechanism.All these have laid the foundation for the research of RSV resistance genes.