Cloning And Functional Analysis Of The Major QTL RppK22 Resistance To Southern Corn Rust And Major QTL Mapping Defence Against Northern Corn Leaf Blight In Maize

Maize foliar diseases are the main diseases in corn production area in our country,such as southern corn rust(SCR)and northern corn leaf blight(NCLB).SCR is one of the devastating and widespread airborne fungal diseases around the world,with a wide range of incidence and serious harm.NCLB is a worldwide disease caused by Setosphaeria turcica.Due to the loss of resistance and races mutants of pathogen,the occurrence of NCLB has become increasing serious.It is still an important foliar disease in the main producing areas that can not be ignored.Maize diseases are not only affect the yield,but also have an influence on quality of maize grain,which is an important factor restricting the sustainable maize productivity.Therefore,development and deployment of resistance maize hybrids is proved to be the most important and effective method to control maize foliar diseases,such as SCR and NCLB.In this study,we constructed two recombinant inbred lines to identify the major QTL RppK22,which was used for map-based cloning and gene function analysis.We identified the major QTL qNCLB5.04 conferring resistance to NCLB and preformed fine mapping for this major QTL.The major results are as follows:1.We constructed two RIL populations,which were derived from susceptible lines DAN340 and BY815 both with the same resistant line K22,for identifying QTL resistant to SCR.Based on the high-density genetic maps and multi-environmental phenotypes,two QTL were identified in DAN340/K22 RIL,while four QTL were detected in K22/BY815 RIL.Both of them shared the major QTL RppK22 on the short arm of chromosome 10.This major QTL was attributed to K22 parent and can respectively explain 68% and 66.8% phenotypic variation in two RILs.The mapping results showed that the resistant parent K22 carrys a major QTL RppK22 conferring resistance to SCR,and this major gene has stable resistance effect in different genetic background.2.Based on map-based cloning strategy,the recombinants were screened from genetic segregation populations containing 3392 individuals.This major QTL was narrowed down between RRD77 and SSR258-3 interval though comparing the genotype of recombinants with the phenotype of its offsprings.Based on the maize B73 reference genome,this region was 16.7 Kb containing two genes,of which Gene1 encodes a CC-NB-LRR protein.3.Three BAC libraries of resistant line K22,susceptible line DAN340 and 1145 were screened.The positive clones covering RppK22 were sequenced by Pac Bio RSII.The BAC sequences were filtered and assembled followed by alignment.As the result showed,RppK22 contains three tandem R genes(R1,R2,R3)in resistance line K22 and one R gene in susceptible lines,DAN340,1145,B73,MO17.There are larger structure variations in promoter and intron of R gene.4.New primers were designed based on the BAC sequences,for sequencing the recombinants and identifying the breakpoints.The recombinations were found in any of three R genes(R1,R2,R3).The R genes of K22 can cross with DR3 gene of DAN340.All disease-resistant recombinant lines carried the intact R3 gene,while the susceptible recombinants contain no R3 gene or an uncomplete R3 gene,indicating that the R3 gene may be the target gene for SCR resistance.24 mutants were screened and identified from 8000 F1 populations derived from the cross between NIL-R and NIL-S.All of those carried the chimeric gene R3-R1,suggesting that R gene clusters are prone to happen unequa crossover.Combined with the result of fine mapping and mutants,it was showed that high frequency recombination among R gene cluster has happened and it happens not only in alleles among different lines,but also within R gene cluters.5.The temporal and spatial expression pattern of RppK22 in resisatant line K22 showed that R2 was not expressed in all tissues while the expression of R3 was detected special in leaf and leaf sheath.The abundance of R3 transcript in leaf was 3 to 4 times higer than that in leaf sheath.The expression pattern of RppK22 before and after SCR pathogen inoculation showed that the expression of R2 gene was not induced by SCR pathogen while the expression level of R3 was not changed after pathogen inoculation.Therefore,R3 is the target gene conferring the resistance to SCR.6.The complementatary result of R3 transgenic plants showed that the resistance of positive plants with R3 allele were significant stronger than that of negative plants in both T2 and F2 segregation population,suggesting that overexpression of R3 CDS could significantly improve resistance to SCR.But the resistance level of transgenic positive plants was not up to that of K22 parent,so we inferred that the R3 CDS sequence could partial restore the resistance to SCR.7.The RppK22 haplotype was analyzed in association mapping panel containing 500 maize inbred lines.17 lines carried the R3 gene,of which 12 had the same haplotype with K22.The R3 haplotype was significantly more resistant than that of none-R3 haplotype.Analysis the frequence of haplotpyes were found that RppK22 gene first was undergone transposon insertion in promoter and intron,following with unequal crossover to form a new haplotype.The K22_like had undergone events,such as duplication and deletion,to generate new haplotype with only R3 or only R2/R3 genes.8.The parent K22 is not only high resistance to SCR,but also high resistance to NCLB.QTL identification was carried out in K22/BY815 RIL by using genetic linkage map and NCLB disease severity level and disease lesion size.Six QTL were identified in both traits.One of them qNCLB5.04 was a co-located QTL,which could explain 19%~25% phenotypic variation in disease severity level.Though fine-mapping strategy using recombinant-derived progeny,qNCLB5.04 was narrowed to 6 Mb region.In brief,RppK22 is a major QTL with large effects and stable resistance to southern corn rust,and high frequency of unequal crossover events are found in this region.The resistance to northern corn leaf blight is a complex quantitative trait,which is under the control of few major genes and many minor genes with relatively low effects.The cloning and dissection of RppK22 and qNCLB5.04 locus will improve our understanding of the molecular mechanism of disease resistance in maize.