Establishment of an experimental evolution protocol. Sexual (SEX) and clonal (CLO) populations of the yeast Schizosaccharomyces japonicus will be propagated in batch cultures in triplicate. A daily serial transfer to YE rich medium will allow fast growth and uniform cell doublings across populations. Meiosis (sporulation) will be induced in sexual lines once every three weeks. Stepwise increments of 0.5 % ethanol throughout the experiment will provide selective pressure to challenge adaptation. Periodic assays will compare relative fitness across populations.                    

Periodic genomic and transcriptomic survey of sexual (SEX) and asexual (CLO) populations. Cells will be sampled from sexual and clonal lines to generate DNA for bulk population genome sequencing. In addition, for each targeted sexual and clonal line, one colony grown on solid medium will be propagated to extract DNA and mRNA for sequencing a unique representative genome and transcriptome. The goal is to uncover genetic variability accumulating in the populations at each sampled point.                                                                                                                                                                                                            

Further study of mutations discovered by use of molecular genetics and genomic tools. Representative DNA mutations found in Aim 2 will be further studied by standard molecular genetics methods to elucidate their possible function in ethanol tolerance. Ad hoc genomic analyses will address the time-course dynamics of DNA variant accumulation.                                                                                                                                                                   

Outreach: the design of a dedicated website. This will serve to communicate the importance of the evolution experiment and to provide educational resources for evolution, and the biology and biotechnology of sexual reproduction.                                                                                                                                                                 

Application: evolution of S. cerevisiae PE-2 under increasing ethanol concentrations. The top industrial fermenter yeast strain Saccharomyces cerevisiae PE-2 will be propagated anaerobically by serial transfers to a medium containing increasing amounts of ethanol. Sporulation (meiosis) will be induced periodically. Samples will be frequently collected for evaluation of specific growth rates, ethanol tolerance, and to be target for genomic/transcriptomic analysis.