Construction Of Mu/MuDr Mutant Library In Maize And Fine Mapping Of LSC1 Gene

Maize is the main food crop,feed and industrial raw material in China.It is widely cultivated in China and is related to the food security of China.Maize Mutator transposons naturally exist in the maize genome.By far,it has been found to have the most active and mutagenic class of transposons.The use of transposons to create mutants is safe and stable compared to other mutagenesis methods such as EMS,and it has a higher mutation frequency.Thus,it is an efficient and reliable method for mutant creation and gene cloning.In this experiment,a transposase activity maize line with MuDR gene and bronze1-mum9gene and a maize line with the Bronze1 gene?anthocyanin synthesis gene?were used.And they were introduced from the genetic background of W22 to the genetic background of B73 through multiple generations backcross.Then the two lines were crossed to obtain the F₁ generation?seed black-purple?,and the F₁ generation was selfed to obtain an F₂ generation which contained a large number of Mu transposon insertion mutation sites.Stable mutant offspring?yellow?were obtained by selecting the color of the F₂ seed Mutant offspring?yellow?were obtained by selecting the color of the F₂ seed.The seeds from these stable mutant progenies selfed constitute the mutant library material that we need.By this method,we screened 183 individuals with obvious phenotypes among 3939 F₂ populations.According to their phenotypic characteristics,they are divided into the following categories:Kernel mutants,Resistance mutants and Plant morphological mutants.The kernel of maize is the most direct reflection of its impact on the yield.Improving the characteristics of the kernel can obtain breeding resources quickly and efficiently.In this study,a big kernel mutant named lsc1?large size corn1?was selected for further research,and the results are as follows:?1?The mutant with larger kernel size was self-crossing for multiple generations until the phenotype was stable.We compared the length,width,thickness,and 100-grain weight of mutants lsc1 and B73.The average grain length of lsc1 was 11.298 mm,and B73 was10.489mm,it is increased by more than 7%.The average grain width of lsc1 is 10.127mm,B73is 6.798mm,which is increased by more than 40%.The average grain thickness of lsc1 is5.968mm,and it is increased by more than 20%compared to 4.803mm of B73.lsc1 100-grain weight was 48.585g,an increase of more than 100%compared to 23.592g of B73.Due to the significant change in grain width,grain width was selected as the target trait of the study.?2?The F₁ generation was obtained by crossing the homozygous lsc1 mutant with the Mo17 inbred line,and the F₂ obtained by F₁ selfing.F₂ population was used as the isolated population for this study.The F₂ generation segregation ratio was counted and found to be in accordance with the Mendel segregation ratio of 3:1.Obviously,this phenotype is controlled by a typical recessive single gene.?3?Screening of 110 pairs of SSR markers,we get 50 pairs of SSR molecular markers that are polymorphically linked between the two parents.It covers 10 chromosomes of the whole genome of maize,and the average distance between markers is about 25Mbps.?4?We counted the grain width of 318 individual plants in the F₂ small segregation population.BSA?Bulked Segregant Analysis?was used to map clone.First,we select the maximum grain width,20 samples of each individual plant and two parents to construct a DNA pool.Then PCR-gel electrophoresis analysis was performed using different SSR primers.Finally,the two molecular markers bnlg2203 and dupssr9 were tightly linked.?5?We expanded the F₂ segregation population,and counted the grain width of each individual plant.Among the 1706 individual plants,400 individual plants with extremely large grain width were selected for fine mapping.Based on the results of the initial localization of the small population,11 available markers were designed and screened near the two molecular markers bnlg2203 and dupssr9 to narrow the candidate interval to 5 Mbps.Moreover,we analyzed the expression patterns and gene functions of the genes in this 5Mbps interval and identified candidate genes.?6?Using Mu flanking sequence enrichment method performed on 50 maximal grain DNA pools and 40 minimum grain DNA pools respectively.And correlation analysis was performed in order to find candidate genes.