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

Abstract

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 (F₀ individuals) can asexually produce unique mutant offspring (F₁) for at least 3 consecutive broods, suggestive of multiple broods as F₁ mutants. Lastly, we estimate that about 750 F₁s are needed for all genes in the Daphnia genome to be mutated at least once with a 95% probability. We also recommend 4-5 F₂s should be collected from each F₁ mutant through sibling crossing so that all induced mutations could appear in the homozygous state in the F₂ population at 70-80% probability.

Fig. 1. Forward genetic approach for obtaining mutant lines in Daphnia.

This study used F₁ mutants to determine the mutation rate and spectrum of EMS-induced heritable mutations.

Fig. 2. Experimental procedure for establishing EMS mutant lines.

A Establishing replicate mutant lines of a Daphnia isolate. B Establishing brood-specific mutant lines of a Daphnia isolate.

Fig. 3. Base-substitution mutation rates of three Daphnia species at 10 and 25 mM EMS treatment.

The bar plot summarizes the species-specific rates based on multiple isolates of each species, whereas the scatter plot represents brood-specific mutation rates in each species.

Fig. 4. Average proportions of different types of base substitutions in each species at 10 and 25 mM EMS treatment.

A Composition of base substitutions in each species. B Composition of base substitutions in different broods.

Fig. 5. Summary of distribution of base substitutions and amino acid changing effects.

Average proportions of base substitutions in different genomic regions (A and B) and amino acid changing effects at 10 and 25 mM EMS concentration (C and D).

Fig. 6. Bars represent the proportion of EMS-induced mutations at each trinucleotide motif centered at mutated sites (5ʹ-3ʹ orientation), whereas the lines represent the observed proportion of trinucleotide frequencies observed in the Daphnia reference assemblies.

NAN and NTN trinucleotides are significantly underrepresented, whereas many of the NGN and NCN motifs are overrepresented (indicated by asterisks).