Fine Mapping Of Two Leaf Mutant Genes Al And Yl In Maize

Mutants are the basic materials for the study of inheritance. Using natural mutants or artificial mutants to clone gene, and then to study biological function of the gene has already become important. Because of its visible phenotype and discernable character, leaf color mutants have been used for researches on chloroplast structure and function, genetic and development, chlorophyll biosynthesis and degradation pathways, molecular mechanisms of photosynthesis and a phenotypic marker used in crop breeding.The leaf mutant was found from natural mutant in the inbred line 444. In previous study, the trait of the leaf mutant 444 was governed by gene al and yl which were mapped on the short arm of chromosome 2 and 7 by SSR analysis, respectively. In this study, we carried out further fine mapping. On the one hand, a big backcross segregating population which was derived from the mutant 444 and the wild-type 9058 had been used to find recombination events by the multiple-PCR method; On the other hand, we developed new molecular markers for further fine mapping of two genes by the use of maize B73 genome sequencing information. Furthermore, candidate genes were predicted on the base of fine mapping with bioinformatics analysis. The brief of results in this study were obtained as follows:1. A backcross population consisting of 9890 individuals was screened for recombination events by two flanking markers bnlg1064 and umc1026 with the strategy of double primers PCR method. 176 recombination plants were identified by bnlg1064 and umc1026, in which 108 were between al and umc1026 and 68 between al and bnlg1064. These recombination plants consisted the group for further fine mapping. Beside, one pair of ILP marker (ILP2-3) and 3 pairs of InDel markers (ID1-2, ID4-1-2 and ID5-8-2) were developed successfully. Consequently, al was flanked by the markers ID1-2 and 2CH10-4, the physical distance between the two markers is about 150 kb.2. By the use of bioinformatics tools to analysis the sequence between the markers ID1-2 and 2CH10-4, it predictes 28 putative genes. After analyzing open reading frames (ORF) and protein sequences of 3 genes, which show high similarity homologous protein to functional genes, the results demonstrated two genes which encode gibberellin 2-beta-dioxygenase-7 and pentatricopeptide (PPR) repeat-containing protein in the mutant 444 and the wild-type 444 exist bases mutation and amino acid changes. Therefore, we will put emphasis on the two genes in further study.3. A backcross population consisting of 1094 individuals was screened for recombination events by two flanking markers umc2160 and 7CH1-5. 161 recombination plants were identified by umc2160 and 7CH1-5., in which 51 were between yl and umc2160 and 110 between yl and 7CH1-5. These recombination plants consisted the population for further fine mapping. 13 pairs of BAC-SSR primers were developed and only 1 pairs of primer (7CH19-4) exhibited polymorphisms between the mutant 444 and the wild-type 9058. Consequently, al was flanked by the markers umc2364 and 7CH19-4, the physical distance between the two markers is about 640 kb, which provided foundation for further fine mapping and candidate gene prediction.