High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed us to start inferring the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. In this work, we generate 32 high-quality reference genomes for the well-known model species D. melanogaster and focus on the identification and analysis of transposable element variation as they are the most common type of structural variant. We show that integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. We also identify hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species. Our results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if we are to understand how genomes function and evolve.

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Most of the current knowledge on the genetic basis of adaptive evolution is based on the analysis of single nucleotide polymorphisms (SNPs). Despite increasing evidence for their causal role, the contribution of structural variants to adaptive evolution remains largely unexplored. In this work, we analyzed the population frequencies of 1,615 Transposable Element (TE) insertions annotated in the reference genome of Drosophila melanogaster , in 91 samples from 60 worldwide natural populations. We identified a set of 300 polymorphic TEs that are present at high population frequencies, and located in genomic regions with high recombination rate, where the efficiency of natural selection is high. The age and the length of these 300 TEs are consistent with relatively young and long insertions reaching high frequencies due to the action of positive selection. Besides, we identified a set of 21 fixed TEs also likely to be adaptive. Indeed, we, and others, found evidence of selection for 84 of these reference TE insertions. The analysis of the genes located nearby these 84 candidate adaptive insertions suggested that the functional response to selection is related with the GO categories of response to stimulus, behavior, and development. We further showed that a subset of the candidate adaptive TEs affects expression of nearby genes, and five of them have already been linked to an ecologically relevant phenotypic effect. Our results provide a more complete understanding of the genetic variation and the fitness-related traits relevant for adaptive evolution. Similar studies should help uncover the importance of TE-induced adaptive mutations in other species as well.

Although transposable elements are an important source of regulatory variation, their genome-wide contribution to the transcriptional regulation of stress-response genes has not been studied yet. Stress is a major aspect of natural selection in the wild, leading to changes in the transcriptional regulation of a variety of genes that are often triggered by one or a few transcription factors. In this work, we take advantage of the wealth of information available for Drosophila melanogaster and humans to analyze the role of transposable elements in six stress regulatory networks: immune, hypoxia, oxidative, xenobiotic, heat shock, and heavy metal. We found that transposable elements were enriched for caudal, dorsal, HSF, and tango binding sites in D. melanogaster and for NFE2L2 binding sites in humans. Taking into account the D. melanogaster population frequencies of transposable elements with predicted binding motifs and/or binding sites, we showed that those containing three or more binding motifs/sites are more likely to be functional. For a representative subset of these TEs, we performed in vivo transgenic reporter assays in different stress conditions. Overall, our results showed that TEs are relevant contributors to the transcriptional regulation of stress-response genes.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington’s disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in aging humans. To address this, we generated induced neurons (iNs) through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. HD-iNs displayed profound deficits in autophagy, characterised by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in ctrl-iNs, highlighting the importance of wild type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington’s disease neurons and provides a new rational for future development of autophagy activation therapies.

18Mapping genotype to phenotype is challenging because of the difficulties in identifying 19 both the traits under selection and the specific genetic variants underlying these traits. Most 20 of the current knowledge of the genetic basis of adaptive evolution is based on the analysis 21 of single nucleotide polymorphisms (SNPs). Despite increasing evidence for their causal 22 role, the contribution of structural variants to adaptive evolution remains largely 23 unexplored. In this work, we analyzed the population frequencies of 1,615 Transposable 24 Element (TE) insertions in 91 samples from 60 worldwide natural populations of 25 Drosophila melanogaster. We identified a set of 300 TEs that are present at high 26 population frequencies, and located in genomic regions with high recombination rate, 27 where the efficiency of natural selection is high. The age and the length of these 300 TEs 28 are consistent with relatively young and long insertions reaching high frequencies due to 29 the action of positive selection. Indeed, we, and others, found evidence of selective sweeps 30 and/or population differentiation for 65 of them. The analysis of the genes located nearby 31 these 65 candidate adaptive insertions suggested that the functional response to selection is 32 related with the GO categories of response to stimulus, behavior, and development. We 33 further showed that a subset of the candidate adaptive TEs affect expression of nearby 34 genes, and five of them have already been linked to an ecologically relevant phenotypic 35 effect. Our results provide a more complete understanding of the genetic variation and the 36 fitness-related traits relevant for adaptive evolution. Similar studies should help uncover the 37 importance of TE-induced adaptive mutations in other species as well.38 39In this work, we screened 303 individual genomes, and 83 pooled samples (containing from 83 30 to 440 chromosomes each) from 60 worldwide natural D. melanogaster populations to 84 identify the TE insertions most likely involved in adaptive evolution (Fig 1). In addition to 85 the age and the size of the 1,615 TEs analyzed, we calculated four different statistics to 86 detect potentially adaptive TEs. The GO enrichment analysis of the genes located nearby 87 our set of candidate adaptive insertions pinpoint response to stimulus, behavior, and 88 development as the traits more likely to be shaped by TE-induced mutations. Consistent 89 with these results, genes located nearby our set of candidate adaptive TEs are significantly 90 enriched for previously identified loci underlying stress-and behavior-related traits. 91Overall, our results suggest a widespread contribution of TEs to adaptive evolution in D. 92 melanogaster and pinpoint relevant traits for adaptation. 93 94 Results 99 Natural populations of D. melanogaster contain hundreds of polymorphic TEs at high 100 population frequencies 101To identify TEs likely to be involved in adaptation, we looked for TEs present at high 102 population frequencies, and located i...

The effect of Medicago sativa (alfalfa) ferritin gene (MsFer) on abiotic stress tolerance was tested using transgenic Vitis berlandieri 9 Vitis rupestris cv. 'Richter 110' grapevine rootstock lines. Leaf discs from transgenic plants maintained higher photosynthetic activity after NaCl, tert-butyl-hydroperoxide (t-BHP) or paraquat treatment than control ones. These results indicate that the increased production of ferritin significantly improved abiotic stress tolerance in transgenic grapevine plants.