Mapping And Cloning Of Two Important Loci In Maize

Drought stress is a major challenge for global agriculture.One of the most important ways to improve the drought resistance of Maize?Zea mays L.?is to reduce plant water loss,and leaf-wilty mutant is a crucial genetic material for leaf water loss study.The research of the biological function and mechanism of leaf wilting mutant gene?s?will improve our understanding of the regulation mechanism of water absorption,transportion and loss in maize,and also will be helpful for the genetics improvement of maize drought resistance.In order to explore the genetic mechanism of the leaf-wilty mutantion,one maize mutant,designated as multi-trait weakened?muw?,which obtained from maize inbred line“Lian 87”was used for the mutant loci mapping and genetic analysis.In addition,kernel row number?KRN?is one of the most important yield related traits in maize.Therefore,the cloning of key candidate gene controlling KRN,exploring of the excellent alleles,and the functional analysis of the key genes will not only help us to clarify the genetic basis of KRN,but also provide the genetic resources and theoretical guidance for the genetic improvement of maize KRN.In this study,a novel major QTL KRN8.03 controlling KRN was fine-mapped and cloned via map-based cloning strategy;the biological function of the key candidate gene was analyzed by expression analysis and candidate gene association analysis etc.The main results obtained in this study were as following:1.Phenotypic analysis of muw.The muw exhibited dehydrated leaf tips and margins even under well-watered field growth conditions.The wilting phenotype is more obvious under high temperature and drought conditions in the field.Compared with the wild-type plants,the growth and yield of muw mutant plants were seriously affected;the water loss rate of the muw leaves was significantly faster than that of the wild-type leaves.In addition,some factors,including stomatal density,cuticle permeability,ABA concentration and ABA sensitivity,vascular bundle and conduit,were identified and compared between muw mutant plants and wild-type plants,and the results showed that only the stomatal density in muw mutant leaves was significantly higher than that of the wild-type leaves(P=1.86×10^-7),however,there were no different in other traits between muw and the wild-type plants.2.Mapping of muw.Genetic analysis showed that muw was controlled by a single recessive locus.The muw mutant locus was located on chromosome 2 between the markers umc1485 and umc1635 by bulked segregation analysis?BSA?method.Then muw was mapped to a 5.66-Mb region between markers V78 and V140.According to the exchange rate of the target region and the results of PCR,we hypothesized that there were large chromosome segment deletion in the muw target region.3.Identification and verification of large fragment deletion.82 primers located in the muw target region were used to do PCR analysis between muw and wild-type plants and confirm the exact position of the deleted segments;combined with the sequencing analysis,a 5,161,714 bp fragment containing 48 genes was finally identified as missing in muw genome.The results of genomic PCR amplification showed that 48 candidate genes could not amplify any products in muw,but could amplify products in wild-type.After that,20 genes which expressed in leaf tissue of wild-type seedlings were found,but no expression was detected in muw leaf tissue;all the results indicated that the 5.16 Mb fragement is indeed lost in the muw genome.4.QTL linkage mapping of KRN and fine mapping of QTL KRN8.03.A total of 12QTLs were detected for KRN under four environments using the V54×Lian87 F_2:3mapping population.Among these QTL,KRN2,KRN4-2 and KRN8.03 were common detected under all four environments and explained more than 10%of the phenotypic variation.Then KRN8.03 was narrowed to a genomic interval of 150 kb between markers M9 and M11using advanced-backcross population and progeny test,and only one gene was predicted in this interval and was designated as KRN8.03.5.Candidate gene association analysis indicated that four SNPs?SNP435,SNP453,SNP493 and SNP528?in a strong linkage disequilibrium?LD?block were significantly associated with KRN.Only SNP493?G/A?caused amino acid missense mutation?Ala/Thr?.Two haplotypes?Hap1 and Hap2?composed four SNPs were identified in 432 maize inbred lines and Hap1 was responsible for increasing KRN.The results of qRT-PCR showed that the expression levels of KRN8.03 were different in the young ear between near-isogenic lines KRN8.03^Lian87ian87 and KRN8.03^V54?P<0.05?.6.Vector construction and genetic transformation.To test the function of candidate gene KRN8.03,the over-expression vector and gene editing vector based on CRIPSR-Cas9 system were constructed.The seeds of T1 for over-expression transformation and T0 seeds for gene editing transformation were obtained.