Loss Of Heterozygosity By SCRaMbLEing

Genetic variation is an important intrinsic factor in species evolution,studying genetic variation is of great significance for revealing evolutionary laws and genetic modification.Loss of heterozygosity refers to the deletion of homologous chromosome fragments in hybrid cells,which causes the alleles to change from heterozygous to homozygous.Due to the cumbersome operation and low efficiency of traditional research methods,the understanding of the loss of heterozygosity is limited.Rapamycin is a macrolide compound that is clinically used to inhibit rejection of organ transplants and it is important to study the resistance of rapamycin.This study used SCRa Mb LE system to explore the association between yeast rapamycin resistance phenotype and loss of heterozygosity.In this study,the genomic rearrangement system was used to drive the rapid evolution of synthetic hybrid yeast strains,and 7 strains with improved rapamycin resistance were obtained.By whole-genome sequencing analysis,loss of heterozygosity of different scales were detected in the evolutionary strains,including short-range loss of heterozygosity,long-range loss of heterozygosity,and whole-chromosomal loss of heterozygosity.The experimental results further revealed that the loss of heterozygosity of gene GLN3 on synthetic V chromosome and the loss of heterozygosity of whole synthetic X chromosome were associated with increased resistance of yeast rapamycin.This study also carried out a comparative analysis of SCRa Mb LE methods and rapid adaptive evolution methods.Using a rapid adaptive evolution method obtained strains with improved resistance to rapamycin,chromosome VIII replication(aneuploidy)was detected by whole-genome sequencing at high frequency,and the results shows that chromosome VIII replication is associated with improved rapamycin resistance phenotype.Continuous subculture experiment indicates that the aneuploid strains shows genomic instability and lead to rapamycin resistance phenotypic degradation.This study deepens the understanding of the genetic mechanism of yeast rapamycin resistance and provides a novel approach to loss of heterozygosity and phenotypic association analysis.