The genome-wide rate and spectrum of EMS-induced heritable mutations in the microcrustacean Daphnia: on the prospect of forward genetics.

Forward genetic screening using the alkylating mutagen ethyl methanesulfonate (EMS) is an effective method for identifying phenotypic mutants of interest, which can be further genetically dissected to pinpoint the causal genetic mutations. An accurate estimate of the rate of EMS-induced heritable mutations is fundamental for determining the mutant sample size of a screening experiment that aims to saturate all the genes in a genome with mutations. This study examines the genome-wide EMS-induced heritable base-substitutions in three species of the freshwater microcrustacean Daphnia to help guide screening experiments. Our results show that the 10 mM EMS treatment induces base substitutions at an average rate of 1.17 × 10(-6)/site/generation across the three species, whereas a significantly higher average mutation rate of 1.75 × 10(-6) occurs at 25 mM. The mutation spectrum of EMS-induced base substitutions at both concentration is dominated by G:C to A:T transitions. Furthermore, we find that female Daphnia exposed to EMS (F(0) individuals) can asexually produce unique mutant offspring (F(1)) for at least 3 consecutive broods, suggestive of multiple broods as F(1) mutants. Lastly, we estimate that about 750 F(1)s are needed for all genes in the Daphnia genome to be mutated at least once with a 95% probability. We also recommend 4-5 F(2)s should be collected from each F(1) mutant through sibling crossing so that all induced mutations could appear in the homozygous state in the F(2) population at 70-80% probability.