Consequences of cryopreservation in diverse natural isolates of Saccharomyces cerevisiae

Experimental evolution allows the observation and assessment of changes over time as laboratory populations evolve to novel, controlled environments. Microbial evolution experiments use cryopreservation to archive experimental populations. Research with Escherichia coli suggests that cryopreservation conditions can affect cell viability and that allele frequencies across the genome can change in response to a freeze/thaw event. We expand on this work by characterizing fitness and genomic consequences of cryopreservation in diploid yeast populations, and by evaluating the effects of multiple consecutive freeze/thaw cycles. Our focal study population is a highly recombinant Saccharomyces cerevisiae strain (SGRP-4X) which harbors standing genetic variation that cryopreservation may threaten. We also investigate the four parental isogenic strains crossed to create the SGRP-4X. We measured cell viability immediately before freezing and after thawing over 5 consecutive cryopreservation cycles. We report evidence that viability increased over time in the SGRP-4X when preserved in both 15% glycerol and 25% glycerol, but we observed no such viability improvements in the parental strains. We also collected genome-wide sequence data from experimental populations initially, after one freeze/thaw, and after five freeze/thaw cycles. In the recombinant SGRP-4X, we find a region of significant allele frequency change on chromosome 15 containing the ALR1 gene. In the parental strains, we find little evidence for new mutations. We conclude that cryopreserving yeast populations with standing genetic variation may have both phenotypic and genomic consequences, though these same cryopreservative practices may have very small effects on populations with little or no initial variation.