A single nucleotide variant in the PPARγ-homolog Eip75B affects fecundity in Drosophila
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Evaluation Summary:
Hoedjes et al. examine how a single nucleotide variant in a regulatory region upstream of the Eip75B gene influences key aspects of life history in Drosophila, using RNAi knockdowns, inbred lines and CRISPR/Cas9 allele replacement at the endogenous locus. This study represents one of the very few examples in animals where the effect of a naturally segregating single nucleotide variant on a complex trait is carefully quantified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Single nucleotide polymorphisms are the most common type of genetic variation, but how these variants contribute to the evolutionary adaptation of complex phenotypes is largely unknown. Experimental evolution and genome-wide association studies have demonstrated that variation in the PPARg-homolog Eip75B is associated with longevity and life-history differences in the fruit fly Drosophila melanogaster . Using RNAi knockdown, we first demonstrate that reduced expression of Eip75B in adults affects lifespan, egg-laying rate and egg volume. We then tested the effect of a naturally occurring SNP variant within a cis-regulatory domain of Eip75B by applying two complementary approaches: a Mendelian randomization approach using lines of the Drosophila Genetic Reference Panel, and allelic replacement using precise CRISPR/Cas9-induced genome editing. Our experiments reveal that this natural polymorphism has a significant pleiotropic effect on fecundity and egg-to-adult viability, but not on longevity or other life-history traits. These results provide a rare functional validation at the nucleotide level and identify a natural allelic variant affecting fitness and life-history adaptation.
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Evaluation Summary:
Hoedjes et al. examine how a single nucleotide variant in a regulatory region upstream of the Eip75B gene influences key aspects of life history in Drosophila, using RNAi knockdowns, inbred lines and CRISPR/Cas9 allele replacement at the endogenous locus. This study represents one of the very few examples in animals where the effect of a naturally segregating single nucleotide variant on a complex trait is carefully quantified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This is an important paper that will be of broad interest. As the authors point out there has been a lot of work in the genetics of complex traits in the last decade-plus, but very few animal studies have actually validated candidate causative variants in a rigorous manner. The human and mouse literature is especially troublesome, so it will be up fly people to do the heavy lifting, although they have not done much yet. Lots of validation efforts exist, but they tend to be correlative and prone to various artifacts. Here the authors attempt to create fly strains that differ at only a single nucleotide, and show that this change has a measurable impact on fecundity (but not some other traits people have posited this nucleotide could impact). The work is rather trailblazing in this regard.
Overall the RNAi …
Reviewer #1 (Public Review):
This is an important paper that will be of broad interest. As the authors point out there has been a lot of work in the genetics of complex traits in the last decade-plus, but very few animal studies have actually validated candidate causative variants in a rigorous manner. The human and mouse literature is especially troublesome, so it will be up fly people to do the heavy lifting, although they have not done much yet. Lots of validation efforts exist, but they tend to be correlative and prone to various artifacts. Here the authors attempt to create fly strains that differ at only a single nucleotide, and show that this change has a measurable impact on fecundity (but not some other traits people have posited this nucleotide could impact). The work is rather trailblazing in this regard.
Overall the RNAi knockdowns only add a little, and the work's real contribution is going to be the gene replacement experiment, and the DGRP crossing experiment. I thought there was a little more work that could be done to make the DGRP crossing experiment more valuable. I think it important to include heterozygous genotypes (is the variant recessive or additive?) and to quantify the contribution of the manipulated variant to total variation. Similarly, I thought the supplement could have described the replacement strategy a little better. It is unclear what the background is in which the replacements are measured? Is it isogenic (that is stated but never shown) ... what is the evidence? Are there other parts of the genome segregating ... can one totally exclude segregating variation as an explanation for the results. I guess this is perhaps an omission and not an error, but a little more validation of the backgrounds is required. Finally, I would have liked to have seen some speculation as to how this particular SNP mechanistically impacts female fecundity.
I hope these concerns are mere speed-bumps for a paper that is otherwise strong. In some cases what was done just has to be explained a little better. In other cases a small amount of sequencing or a few more crosses would greatly strengthen the paper.
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Reviewer #2 (Public Review):
The authors aim to link a SNP to variation in a complex phenotype. This paper builds on considerable prior research on Eip75B and uses three distinct functional genetic methods to measure the phenotypic effects of allelic variation. The work is thorough and, when combined with prior work identifying variation in this locus, represents an unusually complete look at the link between a specific genetic variant and its pleiotropic phenotypic effects.
The major strength of this work is the depth to which variation at this specific locus is investigated. Conducting three separate experiments, each with independent line-level replication within them, is admirable. Together, these data provide a convincing demonstration that Eip75B has effects on egg production. The authors clearly demonstrate that a single SNP can …
Reviewer #2 (Public Review):
The authors aim to link a SNP to variation in a complex phenotype. This paper builds on considerable prior research on Eip75B and uses three distinct functional genetic methods to measure the phenotypic effects of allelic variation. The work is thorough and, when combined with prior work identifying variation in this locus, represents an unusually complete look at the link between a specific genetic variant and its pleiotropic phenotypic effects.
The major strength of this work is the depth to which variation at this specific locus is investigated. Conducting three separate experiments, each with independent line-level replication within them, is admirable. Together, these data provide a convincing demonstration that Eip75B has effects on egg production. The authors clearly demonstrate that a single SNP can have effects on a complex phenotype, albeit not the primary phenotype Eip75B had previously been associated with (lifespan).
This finding is important because it provides validation that GWAS and evolution experiments are capable of locating single nucleotide variants with demonstrable effects on complex traits.A limitation of this work is that the authors do not provide much discussion of the implications of their findings. By necessity, their experiments focus primarily on one of many SNPs in a gene where prior work has detected many different candidate SNPs associated with phenotypic variation in complex traits. It would be good to learn whether future work should systematically functionally characterize all variants, whether this is a worthwhile pursuit for complex traits, and what should be done about those traits that are (nearly) omnigenic. I suspect the findings of this paper will be influential and it could be fruitful for the authors to provide additional perspective for readers seeking to build on it.
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