Etiolation Mutant Gene Mapping Via Bulked Segregant RNA-Seq (BSR-Seq) Method In Maize

Photosynthesis mechanism and development of chloroplast research is undoubtedly a hot issue in biology,while leaf-color mutants were important material for it.In this work, using corn natural mutation YL1001for material,the major aspects of phenotypic traits, chloroplast ultrastructure, chlorophyll content, photosynthetic characteristics and genetic characteristics of mutant were studied and the casual gene was mapped.The main findings are as follows:1. Inbred populations showed the progeny present segregation ratio of3:1(wild type: mutant type=229:83, P>0.05),reciprocal cross also showed mutant trait was not affected by cytoplasmic genetic material, indicating the mutant phenotype controlled by a single nuclear recessive gene. Mutant plants displayed a pale green virescent seedling phenotypes across its whole growth period, and weaker growth potential than wild-type plants, also there is no development of cob and tassel.2. The development of chloroplast was observed by transmission electron microscopy. It show that there is fewer chloroplast in mutant type cell comparison with the wild type, distribution in the cell is also irregular, Starch grains was significantly reduced and have arranged looser thylakoid structure. Compared with the wild type, chlorophyll content of mutant seedling were significantly decreased (P<0.01),chlorochyll a and chlorophyll b are decreased to25.3%and25.7%respectively.Chlorophyll fluorescence parameters were measured and found that there was significantly difference between mutant type and wild type photosynthetic efficiency.3. Use BSR-Seq method to map the mutant gene, two samples were discovered343913SNP loci.Classical Bayesian algorithm was used to SNP loci analysis, and ultimately to identify the casual gene was located in the genomic regions of Chr5:12-14Mb place.4. Developing SSR markers in the BSR-Seq predicted chromosomal segments, detected genotypes in segregating population to verify the results of BSR-Seq. The target zone is mapped in a region of about425.9kb between marker M50020and M50029.Bioinformatics analysis showed that the interval contains17genes,6of which is the chloroplast targeted genes.Wherein the genes GRMZM2G002754which had significantly reduced expression in mutant-type plants and had relationship between leaf color phenotype in its function was deemed as candidate gene.