Genetic Analysis, Fine-mapping And Cloning Of Lfy1Mutant In Maize

Leafy （lfy1） is a dominant mutant in maize that has been shown to particularly increased leaf number above the ear. It has been studied not only for floral transition mechanisms of the maize but also as an important germplasm for silage production in maize. The lfy1locus was reported to be located on the long arm of chromosome3, but few of studies has involved in the molecular cloning of the interested gene. In this study, We isolated a maize lfy1mutant which was remarkably influenced by the genetic backgounds of the inbred lines. We conducted fine mapping of lfy1with lfy1×B73F2-segregated populations, and constructed the physical map for lfy1and followed by the candidate genes confirmation. Also, additional QTLs for leaf number above the ear were detected by lfy1×zheng58F2-segregating population. In addition, two traits, i.e.,the bract number and leaf number above the ear, were also detected via GWAS （Genome-wide Association study） analysis with a natural diverse panel. The main results are as follows:1. lfy1mutant was not completely phenotypically dominant according to the segregation ratio in three F2populations with different genetic background （inbred lines:Zheng58, B73and X178）. For segregated F2population with Zheng58background, the leaf number above the ear exhibited a normal distribution and could be considered as a quantitative trait. While the F2population with B73background segrageted with significant ratio of1:3, which appeared to be controlled by a single dominant gene. Additionally, we use a F2population with B73background as the mapping population for fine mapping of the lfy1gene with newly developed SSR and InDel markers. The lfy1gene was located in a55-kb interval based on the B73reference genome sequence. Three InDel markers were all co-segregated with the leafy phenotypes which provide us molecular markers for genotyping the lfy1mutant related populations and help us for phenotyping the lfy1mutant which shows delayed developmental phenomena on the shoot apical meristem during the seedling stage.2. According to B73reference genome, there are two genes in the mapping interval. We obtained the gene sequences of the two candidates including the transcriptions and the amino acids. There are few polymorphisms detected for gene GRMZM2G072052in lfy1mutant, and the inbred line zheng58also shows the same hyplotype with the lfy1mutant in this gene by compared the sequence with B73reference genome. Also there is no difference detected in transcription of gene GRMZM2G072052. By further study, a5kb transposon was detected in gene GRMZM2G072080in Ifyl mutant which supposed inserted in the intron which do not affect the transcription length of gene GRMZM2G072080in lfy1mutant. Moreover, the RNAi transgenic line of GRMZM2G072080do not show leafy phenotypes. These results above indicate that both genes GRMZM2G072052and GRMZM2G072080may not be the right lfy1gene.3. We constructed the BAC library for lfy1mutant and the Fosmid library for zheng58for screening the causal gene in the mapping interval. Three positive BAC clones detected in this interval were overlapped. Sequenced one of the positive BAC clone named29E3, the sequencing result of the 29E3BAC clone shows significant difference from B73reference genome and gives a longer sequence （>90kb） by blast onto B73genome sequence. Further more, A positive Fosmid clone （26D6） for GRMZM2G072080which detected form zheng58Fosmid library was also sequenced. The26D6clone had a26kb-overlapping region with29E3clone and exactly same sequence with each other. These results show that the lfy1mutant and zheng58were genomic different from the sequenced B73genome, and in this lfy1region, lfy1mutant and zheng58have the exactly same sequence information. And the difference between lfy1mutant and zheng58sequences may be the ture mutantation site of the causal gene.4. For the segregated F2population of zheng58background, the leaf number above the ear which observed as a quantitative trait. The primary QTLs mapping of lfy1was conducted on a F2population with190individuals with110SSR markers, Four QTLs were detected on chromosome2,3,5and10, respectively. The lfy1gene was belong to QTL qEPN3, which can explain the largest12.3%of the phenotypic variance and another QTL qEPN2can explain7.1%of the phenotypic variance. A designed SSR makers （AC209804-4） and SNP maker （chr2_S209011873） were detected in QTLs qEPN3and qEPN2, respectively, was confirmed closely linkage to the corresponding QTLs.5. The leaf number above the ear and the bract number of366inbred lines were investigated separately in years2011and2012. Association analysis of the two traits shows that the correlation co efficiency between the leaf number above the ear and the bract number is0.38. The follow up GWAS （Genome-wide association study） analysis with the55OK SNPs of the366inbred lines on these two traits shows that56SNPs for bract number and45SNPs for leaf number above the ear were detected to be significantly associated at a threshold of p<0.0001.