Mapping, Identifying And Cloning Major Quantitative Trait Genes In Budding Yeast (Saccharomyces Cerevisiae)

Dissecting polygenic variation of complex traits at genic and transcriptional levels is one of the most challenging tasks in the era of functional genomics. The major obstacle has hindered progress in the path from mapping to identifying the genes that underlie quantitative genetic variation: efficacy of the current theoretical and experimental strategies is far from being adequate in dissecting complex genetic architecture at molecular level. The present thesis reports my efforts in exploiting feasibility and challenges in tackling this key problem by taking power of the classical quantitative genetic analysis and the modern genetical genomics.We took ethanol tolerance in budding yeast as a simple model of quantitative traits. The trait phenotype was scored as the critical concentration of ethanol in culture medium at which the test strain or individual can survive in its log-growth phase. Appropriateness of the phenotype measurement was experimentally tested and verified. Quantitative genetic analysis with the trait provided basic genetic parameters and inheritance model of genes contributing variation of the trait. It revealed that as high as 55% proportion of trait phenotype variation can be explained by additive genetic variance.We established a two-way recurrent selection and backcross breeding scheme, whose power in precision and high-resolution mapping of quantitative trait loci (QTL) has been theoretically proved, to create mapping population in order to locate the QTL. The mapping was also carried out in a conventional F₂ segregating population. The RSB based QTL mapping approach was superior to the segregating population analysis in its robustness and accuracy in locating the candidate QTL genes and in its resolving power in pinpointing the most likely locations of the QTL genes. Base on the map information, we were able to narrow the genome region that showed extremely significant linkage with the trait phenotype down to 10 kb. Genetical analysis focusing on this small region has refined and finally identified location of one major QTL gene candidate. Sequencing analysis of this gene revealed large differencein coding sequence between the two parental inherited alleles. The promoter region of the gene and its cw-regulatory motifs were determined and assessed in light of their effect on gene expression. The allele-specific effect on the trait phenotype was confirmed by experimental assay based on gene knock-out and gene replacement.