Study On The Rearrangement Of Ⅴ Chromosome In Triploid Saccharomyces Cerevisiae

Polyploidy endows species with long-term evolutionary flexibility and plays a creative role in the process of genome variation.Aneuploidy and mitotic homologous recombination are common chromosomal variations in polyploid yeast cells,which are characterized by chromosome deletion and a large number of genome rearrangements.It is a huge challenge to construct a polyploid yeast strain that can artificially induce rearrangement and visually track its evolution.In this study,synthetic polyploid Saccharomyces cerevisiae cells were used as a model,and SCRa Mb LE(Synthetic Chromosome Rearrangement and Modification by Lox P-mediated Evolution)genome rearrangement system was used to accelerate the evolution of polyploid Saccharomyces cerevisiae carrying synthetic chromosomes and build a triple yeast genome rearrangement library.PCRTag was used to detect the existence of V chromosome in the genomic rearrangement strain,and the analysis found that the wild-type V chromosome was completely exist.The synthetic V chromosome mainly presents three different genotypes: complete deletion,partial deletion and complete existence,indicating that the synthetic triploid yeast genome is more likely to produce genotype diversity after rearrangement.Based on PCRTag,a method for detecting the copy number of homologous chromosomes was developed to analyze the variation events of synthetic V chromosomes and homologous wild-type V chromosomes in polyploid cells.In the selected 274 wild-type adaptive SCRa Mb LEant libraries,it was found that the synthetic V chromosome deletion phenomenon was more obvious,the frequency was as high as71.53%,and the corresponding homologous wild-type V chromosome was duplicated,resulting in 3n-1 type aneuploid cells.Using the CRISPR-Cas9 method to controllably delete the synthetic V chromosome and artificially construct aneuploid strains,it proved that the aneuploidy phenomenon in triploid yeast cells can exist stably.And through a series of phenotypic characterization methods,it is confirmed that the deletion of synthetic V chromosome will not affect the normal growth of cells,but will only slow down the growth rate of some cells and change cell morphology.In addition,this study also established a method for analyzing mitotic recombination to detect chromosome V recombination events that occurred in the polyploidy reorganization library.The study found that the 118 kb～160 kb of chromosome V is the hotspot of mitotic homologous recombination.These results indicate that SCRa Mb LE provides a powerful platform for cells to produce interchromosomal or intrachromosomal rearrangements,and promotes the understanding of the evolutionary process of polyploid yeast cells.Polyploid Saccharomyces cerevisiae cells drive genome evolution through chromosome deletion or mitotic recombination.By analyzing and drawing high-resolution whole-genome sequencing maps,rapid and systematic identification of the evolution of chromosomes can be achieved,which is expected to provide a good biological model for studying cancer genome changes.