EMS Mutagenesis In Maize With Gene Mapping And Characterization For Colorless Mutant And Kernel Mutants

Maize is one of the leading crops,serving widely as industry material,animal feed and human food.Moreover,maize has served long-time as the model plant for the study in genetics and development.Mutants are valuable tools for maize germplasm innovation,gene discovery and function characterization.Common methods for maize mutagenesis include transposon system,physical and chemical treatment.Compared to other methods,EMS(ethy methylsulfone)has several advantages,such as,most of the mutations are SNPs,mutation sites are randomly distributed,etc.Recently,advances in high-throughput sequencing enabled the quick mapping of EMS mutants.In this study,surrounding creation of maize mutants and functional gene characterization,we performed the following studies:1.Through EMS mutagenesis of maize pollen,we created a series of mutants for inbred line B73 with very rich phenotypic variations observed,including defective in kernel,photosystem defective,dwarf and other special-type mutants.2.Comprehensive studies were performed on a colorless mutant,including gene mapping and molecular characterization.This mutant showed decreased level of pigmentation throughout the whole-life cycle and in multiple tissues.Genetics analysis indicated that the mutant phenotype was controlled by a single recessive allele.Using Mut Map strategy,p.Glu183 Lys mutation(missense)in maize chalcone synthase C2(ZmC2)was identified as the causal.The Loss-of-function of ZmC2 with E183 K mutation was further validated through transformation in Arabidopsis mutant with TDNA insertion in the homologous gene of ZmC2.We evaluated transcriptomic and metabolic changes in maize first sheaths caused by the mutation.RNA-Seq showed that very few genes changed their expression pattern.Metabolome showed that the downstream biosynthesis including flavonoid biosynthesis was blocked by the mutation,while the synthesis of scopolin may be the major shunt in the upstream phenylpropanoid biosynthesis.ZmC2-E183 site is highly conserved in chalcone synthase among Plantae kingdom(evolution)and within species’ different varieties(domestication).Through prokaryotic expression,transient expression in maize leaf protoplasts and stable expression in Arabidopsis,we observed that E183 K and other mutations on E183 could cause almost complete protein aggregation of chalcone synthase.Based on structure analysis,a proposed protein aggregation model was raised with further validation.Combing pollen-mediated transformation and ZmC2 gene,we raised a quick screening strategy for maize transformation.3.We gave the detail of genotypic changes along with EMS mutagenesis for heritable kernel mutants,and raised the quick mapping strategy.For two shrunken kernel mutants,we identified p.Trp186* and p.Pro155 Leu mutation in the same gene ZmBT1,which encoding adenine nucleotide transporter and was responsible to transport ADP-glucose from cytosol to amyloplast.Thus,the mutation interrupted starch biosynthesis in maize endosperm,leading to the shrunken phenotype.For a dent kernel mutant,we identified p.Gln645* mutation in gene Zm SBE2,which encoding a starch branching enzyme and catalyzing the transformation of-1,4-glucan to-1,4/-1,6-glucan.The identified mutation caused the interruption of amylopectin synthesis in maize endosperm,leading to the dent phenotype.For a small and dark kernel mutant,two candidates were identified.Of them,Zm UBP5(encoding ubiquitin-specific protease)with p.Gln458* mutation was more likely to be the causal after analysis.In consistence,mutant-type kernels had reduced level of carotenoids and looked paleyellow from the appearance.In summary,we created a series of maize mutants and identified key genes and mutations for colorless mutant and several kernel mutants related to important agronomic traits.Our study would provide foundations for the exploration of regulatory network in maize growth and development,and for further germplasm innovation and genetic improvement in maize.