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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - DROSADAPTATION (New approaches to long-standing questions: adaptation in Drosophila)

Teaser

Summary

Our project aims at understanding how organisms adapt to their environment. Adaptation is one of the central evolutionary processes. However adaptation has implications that extend well beyond the evolutionary biology field. This is so because adaptation underlies such important processes as the ability of species to survive in changing environments, host-pathogen interactions, and resistance to pesticides and drugs (e.g. antibiotics or cancer chemotherapies). Thus understanding adaptation is important for society as it has consequences for conservation biology, medicine, or agriculture.
Understanding adaptation requires not only identifying the underlying mutations, but also elucidating the molecular mechanisms behind these changes, and the organismal traits affected by the adaptive mutations. These are the three overall objectives of this grant. To accomplish these objectives, we are using state-of-the-art techniques, such as Oxford Nanopore sequencing technology and CRISPR/Cas9 in the model organism Drosophila melanogaster.

Work performed

We have made progress towards achieving our three main objectives. Our first objective is to identify adaptive transposable element (TE) insertions in Drosophila melanogaster natural populations worldwide. We are currently sampling natural populations of D. melanogaster on a continent-wide scale in collaboration with the European Drosophila Population Genomics Consortium. I have co-founded and I am co-leading this consortium that brings together 61 research groups from 29 countries. The consortium was founded in 2013 and has collected flies yearly since 2014. To date, we have sequenced 169 samples collected in three consecutive years and we are currently in our 5th consecutive collection year.
Our research group is coordinating the sampling in Spain, and it is also in charge of the collections in arid regions. Collecting flies in natural populations is both costly and time-consuming. Thus, we are currently expanding our sampling in arid regions by collaborating with local high schools. This also gives us the opportunity to involve teachers, students, and the population from Spanish rural areas in an European project.
We have successfully established in our laboratory the Oxford Nanopore sequencing technology. This technology allows to generate new reference genomes in a cost- and time- effective manner. So far, we have generated a total of 5 new reference genomes. Note that, only one D. melanogaster reference genome is available to date precluding the analysis of structural variants, specially transposable elements which is the focus of our grant.
The successful implementation of this technique in our laboratory allow us now to generate a whole genome sequence in 2.5 days, from library preparation to obtaining the reads. After troubleshooting different aspects of the protocol, we are now able to obtain very high yields, up to 15.6 GB per flow cell, with N50 read length of up to 14.1 kb, proving that this is a very cost-effective technique.

To identify adaptive TEs, it is necessary to estimate the frequency of this type of mutations in populations. We have updated the Tlex2 software that allows to estimate population frequencies. Using all the D. melanogaster samples available to date, including the 2014 collection of the DrosEU consortium, we have already showed that (i) there is no population structure in Western Europe suggesting that it is a good system to identify adaptive mutations (Mateo et al 2018, bioRxiv); ((ii) there is longitudinal structure across the whole European continent (Kapun et al 2018); (iii) there are at least 300 adaptive TE insertions in worldwide populations, a much higher number than the one previously identified (Rech et al in preparation); and (iv) TEs show significant geographical patterns in Europe that are replicated in North American populations (Lerat et al in preparation). Overall, our results so far clearly indicate that TEs play a relevant role in adaptation in natural populations

The second objective of our project is to ascertain the biological mechanisms of adaptation. To accomplish this objective is crucial to analyze both natural strains and mutant strains. We have successfully establish in our laboratory the CRISPR/Cas9 genome editing technique in wild-type strains. Obtaining CRISPR/Cas9 mutants without using transgenic Cas9 flies, and thus preserving the genetic background where the mutation we want to study arised, is very challenging. Indeed, only one CRISPR/Cas9 mutant has been reported using natural strains of D. melanogaster. So far, we have been able to establish two CRISPR/Cas9 mutants. Besides, we have also generated transgenic flies to perform in vivo enhancer assays for 14 TE insertions.
We have also performed an in depth investigation of 14 candidate adaptive TEs located nearby immune-related genes. Allele specific expression analysis showed that 12 out of the 14 TEs affect the expression of their nearby gene. Furthermore, we were able to pinpoint the molecular mecha

Final results

We are generating new reference genomes that will allow us to understand the role of structural variants in their ecological context.
To date, there is a single reference genome available for the model species D. melanogaster. This is limiting our understanding of the role of structural variants in genome evolution as short-read technologies, currently being used by the research community, cannot be used to annotate these variants. We are generating new high quality reference genomes for strains collected in European regions with contrasting climates with the ultimate goal of identifying TE insertions in their ecological context. Generating new reference genomes requires using the newest long-read technologies and having access to High Performance Computing (HPC) cluster. We have already generated five new reference genomes, we are working on eight other genomes, and we are planning to generate more reference genomes to inform our phenotypic assays.

We have successfully established in our laboratory the CRISPR/Cas9 genome editing technique in wild-type strains. Functional validation of adaptive TE insertions required both showing that the TE is associated with a given phenotype in wild-type strains and also generating mutant strains in which the causative TE is deleted from the genome. Generating a mutant strain that only differs from the original wild-type strain by the deletion of a single TE is challenging. CRISPR/Cas9 mutants are often generated either in cell lines or by crossing the strain of interest with a transgenic strain containing Cas9. By crossing the strain of interest to a transgenic strain with Cas9, we would generate an hybrid strain that has a genetic background completely different from the genetic background of the wild-type strain. In our laboratory, we have already successfully establish two strains that differ from the wild-type strain only by the deletion of the TE of interest. We are currently working on the generation of three other CRISPR/Cas9 mutants and we are planning to generate several more before the end of the project.

We are making progress towards the identification of adaptive TE insertions. So far, based on the analysis of worldwide samples including the DrosEU 2014 collection samples we have identified 300 candidate adaptive TEs. We are currently using BayPass to identify the geographic and environmental variables that could be driving the selection on these TE insertions. BayPass also looks for evidence of population differentiation, which potentially could also led to the identification new adaptive TE insertion. We are planning to run BayPass with all available samples that have been collected in the same geographical location in at least two consecutive years.

We are also currently investigating the role of TEs in three ecologically relevant traits: tolerance to the insecticide malathion, tolerance to desiccation, and tolerance to the essential heavy-metal copper. We are planning to perform RNA-seq, ChIP-seq with histone marks, ChIP-nexus for the Cnc transcription factor, ATAC-seq and Hi-C to have a comprehensive view of the mechanisms by which candidate TEs affect these three ecologically relevant traits. CRISPR/Cas9 mutants of the candidate adaptive TEs identified will allow us to establish causal links between the mutations and the fitness effects.