Fine Mapping And Cloning Of Resistance Gene To Northern Corn Leaf Blight In Tropical Maize Germplasm CML493

Germplasm of tropical maize has excellent resistance genes,which can broaden the germplasm base of temperate regions,and created new germplasm groups of maize,thus realizing the construction of new heterosis patterns.The introduction and utilization of germplasm of tropical maize is a long-term development strategies of maize breeding in China and even in the world.There have been many reports on resistance genes of northern corn leaf blight.However,the discovery of resistance genes were located mostly on the basis of primary.In this study,tropical maize germplasm CML493,temperate maize inbred line PH4CV and isolated populations were used as experimental materials.Through the combined analysis of Seq-BSA,Kasp genotyping and transcriptome sequencing technology,combined with the identification of artificial inoculation of setosphaeria turcica fluid,fine mapping and genetic transformation of maize northern corn leaf blight resistance genes.The main research results are as follows:Seq-BSA technology were used to mapping the northern corn leaf blight of tropical maize germplasm CML493.Candidate regions of SNP and In Del correlation area were analyzed,resistance gene of northern corn leaf blight were located on chromosome 8（154240000-168290000）.1685 genes associated area candidate regions and regions length were 14.05 Mb,405 non-synonymous mutations and 97 frameshift mutations.The candidate interval were larger by Seq-BSA technique mapping,KASP marker genotyping technique was used to fine mapping the genes of resistance to northern corn leaf blight in tropical germplasm CML493.SNP primers were designed in the candidate region to reduce the candidate region.Diallele were judged by SNP of specific locus to realize fine mapping resistance gene to northern corn leaf blight in tropical maize germplasm.11 candidate genes were identified by KASP technique between Zm00001d011652 and Zm00001d011662.Different expression of CML493 and PH4CV were analyzed by transcriptome sequencing.Gene expression of 2 materials showed a trend of up-regulated after artificial inocuting setosphaeria turcica.There were 723 coexpressed genes in CML493 and 1038 coexpressed genes in PH4CV.89 and 110 co-expressed genes distributed on chromosome 8 of CML493 and PH4CV.4 co-expression genes of CML493 and PH4CV in the region 154240000-168290000 on chromosome 8.Genes are MKKK18,CDPK21,HEX9 and WAK RLK1.MKKK18 were located in candidate region by KASP technique.Conjoint analysis by Seq-BSA,KASP genotyping,transcriptome sequencing analysis and real-time quantitative PCR technology were used to verify the candidate gene,finally ZmMKKK18 was identified as the candidate gene for resistance to northern corn leaf blight.ZmMKKK18 gene transcribed and encoded 479 amino acids,with theoretical isoelectric point（Pi）of 4.81,estimated molecular weight of 50159.62Da,molecular formula of C₂₁₇₂H₃₄₆₅N₆₃₃O₆₈₆S₂₃,theoretical half-life of 30h,instability parameter of 49.84,it was an unstable protein.Amino acid sequences of ZmMKKK18 and Sb MKKK18 were closely related by phylogenetic tree analysis.Cloning and Functional verification of ZmMKKK18 gene by transgenic technology.A 1645bp full-length ZmMKKK18 gene was cloned and an overexpression vector p EGOEPubi-B-35S-ZmMKKK18 was constructed with a full-length of 12125bp.Bar gene expressed herbicide resistance in plants.Over-expression vector was transformed into maize inbred line PH4CV by non-tissue culture ultrasonic assisted mediated pollen treatment.T₃ generation positive transgenic materials were detected by PCR,and all tested plants obtained the target gene bands for PCR amplification.The relative expression level of ZmMKKK18 gene in positive plants was significantly higher than wild type control by q RT-PCR.The transgenic plants were all positive by Bar test strip.The results showed that both ZmMKKK18 and marker gene fragment were integrated into the T₃generation lines,could be inherited and expressed stably.T₃ transgenic ZmMKKK18gene and wild-type PH4CV plants were inocuted setosphaeria turcica fluid to identify resistance.The transgenic ZmMKKK18 plants had a small amount of spots on the leaf and identified as level 5 resistance.However,the disease resistance of PH4CV was significantly lower than CML493.Wild type PH4CV plant leaf disease spot were severe and identified as level 9 resistance.