Fine Mapping And Candidate Gene Analysis Of A Small Brown Planthopper Resistance Gene QSBPH5

Rice(Oryza saliva L.)is an important grain crop.Maintaining rice production is of great significance to Chinese food security.However,diseases and insect pests are important factors affecting rice yield and quality in the rice production process.rice planthopper(Rice Planthopper)is the most serious destructive insects in rice production,and Small brown p lanthopper(SBPH)has a wide range of host plants.Small brown planthopper can not only distruct rice,wheat and other gramineous crops,by the means of sucking the phloem sap cause yellow leaves and plant death,but also is a natural host for a variety of rice virus diseases,including rice stripe virus(RSV)and rice black-streaked dwarf virus(RBSDV),which can caused Rice Stripe Virus Disease,Rice Black-streaked Dwarf Virus Disease,Wheat Rosette Stunt Virus disease and Maize Rough Dwarf Disease etc.However,the control of SBPH and its transmitted virus diseases still depends on chemical pesticides.due to the large number of small brown planthoppers,their strong migration ability,and the transient transmission of virus,the control effect is poor.At the same time,the application of chemical pesticides increases the cost of agriculture,pollutes the environment,kills natural enemies of SBPH,destroys the ecological balance of farmland,and induces increased drug resistance.At present,the best way to control SBPH and its transmission of viral diseases is to select and breed insect-resistant varieties.It is the premise and basis of cultivating novel resistant varieties to screen resistance resources and mapping and cloning of resistant genes.In this study,NU3 which is highly resistant to SBPH is a family in N22/USSR5 recombinant inbred lines.The resistance gene to SBPH was mapped and some functions were explored.The main results are as follows:1.Resistant cultivar NU3 high resistance to SBPH.At 14 days post infestation with SBPH,the seedling mortality rate of the susceptible cultivar USSR5 was 100%,while the seedling mortality rate of the resistant cultivar NU3 was only 24.24%.At the same time,the seedling mortality rate of the popularized variety Ning jing 7(W030)was 75.5%.The results show that NU3 is highly resistant to SBPH,while USSR5 is highly susceptible to SBPH.We can use NU3 as a source of resistance to mine resistance genes..2.The evaluation of resistance mechanism of NU3In order to reveal the genetic basis of resistance to SBPH in NU3,a genetic segregation population was constructed with NU3 and USSR5.Using Modified seedbox screening test,181 F2:3 lines were identified for resistance to SBPH,and 112 lines with good data repeatability(repetition error<15%)were selected for genetic analysis of resistance to SBPH.The phenotypic data of these pedigrees were continuously distributed from level 0 to 9,and the phenotypic separation ratio was 49:41:22.The result of χ2 test does not match of segregation ratio of Mendel’s regulavity of segregation,indicating that The phenotype of NU3 resistance to SBPH may be controlled by multiple genes.3.The mapping of SBPH-resistant gene with NU3/USSR5 F2:3 populationUsing 11 extreme insect-resistant and 11 extreme insect-susceptible families,an initial linkage analysis was conducted,and signs of linkage were found on the two chromosomes 4 and 5,respectively,and a linkage map of chromosomes 4 and 5 was further constructed.Further,Using the constructed genetic map and combining the phenotypes of 112 F2:3 families,the SBPH-resistance QTL analysis was performed.Two SBPH-resistant QTL loci qSBPH4 and qSBPH5 were detected on two chromosomes 4 and 5,which were located between the markers RM16335～RM6659 and RM405～RM169.The LOD values were 2.5 and 3.5,and the contribution rate was 14.2%And 14.3%.4.The fine-mapping of qSBPH5 and the analysis of candidate genesTo further fine-mapping qSBPH5,we select F2:3 families from NU3/USSR5 to select single plants containing only qSBPH5 loci and heterozygous genotypes.Then screening of recombined individual plants by self-breed to generate F3:4,F4:5 and F5:6 population,and developing continuously encrypted molecular markers.Finally qSBPH5 was fine-mapping to a region of the 80 kb interval.Then combining the results of genome sequencing and SBPH-induced expression,two candidate genes,LOC_Os05g09480 and LOC_Os05g09520,were identified.Transgenic validation was performed on these two genes.5.Auxin negatively regulates SBPH-resistanceThe predicted gene ORF3 is OsIAA16,and it is a suppressor of auxin signal.Exogenous application of auxin and mutant of endogenous increase of auxin can reduce the resistance of SBPH to rice varieties.At the same time,two mutants osarf2 and osarfl4,wihch are auxin downstream response genes,had increased resistance to SBPH.In summary,auxin negatively regulates the resistance to SBPH in rice.6.Calmodulin binding protein participates in resistance to SBPHThe predicted gene ORF7 encodes calmodulin binding protein.Knocking out ORF7 in insect-resistant varieties significantly reduced their resistance to SBPH,and overexpression or transfer of insect-resistant alleles into susceptible varieties significantly increased resistance to SBPH.Studies have shown that ORF7 regulates rice grain width by positively regulating BR signal.To this end,we carried out resistance identification of the rice planthopper in OsGSK2 and OsDLT transgenic families of BR downstream signal-related genes,and found that the BR signaling pathway negatively regulated resistance to SBPH.The above results indicate that ORF7 may regulate the resistance to the planthopper by other means,unlike the mechanism regulating grain width.After that,the interaction protein of ORF7 was screened by IP-MS,and several candidate interaction proteins were obtained.The fine-mapping of the above-mentioned SBPH-resistant gene lays a foundation for the cloning and mechanism of SBPH-resistant.Moreover,it can provide new materials for the marker-assisted selection breeding.