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[EN] Mobile genetic elements are genetic material with the ability to move within the genome or, in some cases, between different organisms or cells. Two classes of mobile genetic elements can be distinguished, viruses, which have the ability to transfer their genetic material between organisms, and transposons, which move and replicate within the genome of the host organism. Transposons occupy an important fraction of eukaryotic genomes and through their movement can alter their structure, playing a key role in the evolution of genomes. In this thesis we have studied the dynamics and impact of Physcomitrium patens of different mobile elements. The first chapter focuses on the analysis and comparison of different methodologies to detect transcription and mobilization of transposons using sequencing data based on short-reads technologies. This analysis allowed us to define the methodologies that best fit the objectives of this thesis. In the second chapter, these selected methodologies were used to study transposon dynamics in the genome of Physcomitrium patens, detecting several families of retrotransposons and DNA transposons that are transcriptionally active and, in some cases, polymorphic in the population. The third chapter describes the analysis of the impact of transposons, and in particular of a retrotransposon with LTRs named RLG1, on both the genome structure and genes of Physcomitrium patens, eliminating or in some cases, reintroducing RLG1 elements at specific locations in the genome. Finally, the last chapter focuses on the detection and dynamics of the first virus described in P. patens. This virus, which we have named Physcomitrium patens Amalgavirus 1 (PPAV1), is an endogenous virus present only in some P. patens accessions and transmitted vertically, both from the paternal and maternal lines., [ES] Los elementos genéticos móviles son material genético con la capacidad de moverse en el genoma o, en algunos casos, entre diferentes organismos o células. Pueden distinguirse dos clases de elementos genéticos móviles, los virus, que tienen la capacidad de transferir su material génico entre organismos, y los transposones, que se mueven y replican dentro del genoma del organismo huésped. Los transposones ocupan una fracción importante de los genomas eucariotas y mediante su movimiento pueden alterar su estructura, teniendo un papel clave en la evolución de los genomas. En esta tesis se ha estudiado la dinámica y el impacto de Physcomitrium patens de diferentes elementos móviles. El primer capitulo se centra en el análisis y la comparación de diferentes metodologías para detectar la transcripción y movilización de los transposones utilizando datos de secuenciación basados en tecnologías de short-reads. Este análisis nos permitió definir las metodologías que mejor se adaptan a los objetivos de esta tesis. En el segundo capítulo se utilizaron estas metodologías seleccionadas con la finalidad de estudiar la dinámica de los transposones en el genoma de Physcomitrium patens, detectando varias familias de retrotransposones y transposones de ADN que son transcripcionalmente activas y que en algunos casos son polimórficas en la población. En el tercer capítulo se describe el análisis del impacto de los transposones, y en concreto de un retrotransposón con LTRs nombrado RLG1, tanto en la estructura del genoma como en los genes de Physcomitrium patens, eliminando o en algunos casos, introduciendo de nuevo elementos RLG1 en lugares concretos del genoma. Finalmente, el último capítulo se centra en la detección y dinámica del primer virus descrito en P. patens. Este virus, que hemos nombrado Physcomitrium patens Amalgavirus 1 (PPAV1) es un virus endógeno presente únicamente en algunas accesiones de P. patens y que se transmite verticalmente, tanto por la línea paterna como ma, [CA] Els elements genètics mòbils son material genètic amb la capacitat de moure's en el genoma o, en alguns casos, entre diferents organismes o cèl·lules. Podem distingir dues classes d'elements genètics mòbils, els virus, que tenen la capacitat de transferir el seu material gènic entre organismes, i els transposons, que es mouen i es repliquen dins el genoma de l'hoste. Els transposons ocupen una fracció important dels genomes eucariotes i mitjançant el seu moviment poden alterar-ne la seva estructura, tenint un paper clau en l'evolució dels genomes. En aquesta tesi s'ha estudiat la dinàmica i l'impacte en Physcomitrium patens de diferents elements mòbils. El primer capítol es centra en l'anàlisi i la comparació de diferents metodologies per tal de detectar la transcripció i mobilització dels transposons utilitzant dades de seqüenciació basades en tecnologies de short-reads, Aquesta anàlisi ens va permetre definir les metodologies que millor s'adapten als objectius d'aquesta tesi. En el segon capítol es van emprar les metodologies seleccionades amb la finalitat d'estudiar la dinàmica dels transposons en el genoma de Physcomitrium patens, detectant varies famílies de retrotransposons i transposons d'ADN que son transcripcionalment actives i que en alguns casos son polimòrfiques a la població. En el tercer capítol es descriu l'anàlisi de l'impacte dels transposons, i en concret d'un retrotransposó amb LTRs anomenat RLG1, tant en l'estructura del genoma com en els gens de Physcomitrium patens, eliminant o, en algun cas, introduint de nou elements RLG1 en llocs concrets del genoma. Finalment, el darrer capítol es centra en la detecció i dinàmica del primer virus descrit a P. patens. Aquest virus, al que hem anomenat Physcomitrium patens Amalagavirus 1 (PPAV1) és un virus endogen present només en algunes accessions de P. patens i que es transmet verticalment, tant per la línia paterna com materna.

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of P. patens, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus–host coevolution.

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation., CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens., Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens., These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.

Transposable elements (TEs) are a rich source of genetic variability. Among TEs, miniature inverted-repeat TEs (MITEs) are of particular interest as they are present in high copy numbers in plant genomes and are closely associated with genes. MITEs are deletion derivatives of class II transposons, and can be mobilized by the transposases encoded by the latter through a typical cut-and-paste mechanism. However, MITEs are typically present at much higher copy numbers than class II transposons. We present here an analysis of 103 109 transposon insertion polymorphisms (TIPs) in 738 Oryza sativa genomes representing the main rice population groups. We show that an important fraction of MITE insertions has been fixed in rice concomitantly with its domestication. However, another fraction of MITE insertions is present at low frequencies. We performed MITE TIP-genome-wide association studies (TIP-GWAS) to study the impact of these elements on agronomically important traits and found that these elements uncover more trait associations than single nucleotide polymorphisms (SNPs) on important phenotypes such as grain width. Finally, using SNP-GWAS and TIP-GWAS we provide evidence of the replicative amplification of MITEs.

Similarly to other plant genomes of similar size, more than half of the genome of P. patens is covered by Transposable Elements (TEs). However, the composition and distribution of P. patens TEs is quite peculiar, with Long Terminal Repeat (LTR)-retrotransposons, which form patches of TE-rich regions interleaved with gene-rich regions, accounting for the vast majority of the TE space. We have already shown that RLG1, the most abundant TE in P. patens, is expressed in non-stressed protonema tissue. Here we present a non-targeted analysis of the TE expression based on RNA-Seq data and confirmed by qRT-PCR analyses that shows that, at least four LTR-RTs (RLG1, RLG2, RLC4 and tRLC5) and one DNA transposon (PpTc2) are expressed in P. patens. These TEs are expressed during development or under stresses that P. patens frequently faces, such as dehydratation/rehydratation stresses, suggesting that TEs have ample possibilities to transpose during P. patens life cycle. Indeed, an analysis of the TE polymorphisms among four different P. patens accessions shows that different TE families have recently transposed in this species and have generated genetic variability that may have phenotypic consequences, as a fraction of the TE polymorphisms are within or close to genes. Among the transcribed and mobile TEs, tRLC5 is particularly interesting as it concentrates in a single position per chromosome that could coincide with the centromere, and its expression is specifically induced in young sporophyte, where meiosis takes place.

[Motivation] Transposable elements (TEs) constitute a significant proportion of the majority of genomes sequenced to date. TEs are responsible for a considerable fraction of the genetic variation within and among species. Accurate genotyping of TEs in genomes is therefore crucial for a complete identification of the genetic differences among individuals, populations and species., [Results] In this work, we present a new version of T-lex, a computational pipeline that accurately genotypes and estimates the population frequencies of reference TE insertions using short-read high-throughput sequencing data. In this new version, we have re-designed the T-lex algorithm to integrate the BWA-MEM short-read aligner, which is one of the most accurate short-read mappers and can be launched on longer short-reads (e.g. reads >150 bp). We have added new filtering steps to increase the accuracy of the genotyping, and new parameters that allow the user to control both the minimum and maximum number of reads, and the minimum number of strains to genotype a TE insertion. We also showed for the first time that T-lex3 provides accurate TE calls in a plant genome., [Availability and implementation] To test the accuracy of T-lex3, we called 1630 individual TE insertions in Drosophila melanogaster, 1600 individual TE insertions in humans, and 3067 individual TE insertions in the rice genome. We showed that this new version of T-lex is a broadly applicable and accurate tool for genotyping and estimating TE frequencies in organisms with different genome sizes and different TE contents. T-lex3 is available at Github: https://github.com/GonzalezLab/T-lex3.

[Background] Transposable elements (TEs) are an important source of genomic variability in eukaryotic genomes. Their activity impacts genome architecture and gene expression and can lead to drastic phenotypic changes. Therefore, identifying TE polymorphisms is key to better understand the link between genotype and phenotype. However, most genotype-to-phenotype analyses have concentrated on single nucleotide polymorphisms as they are easier to reliable detect using short-read data. Many bioinformatic tools have been developed to identify transposon insertions from resequencing data using short reads. Nevertheless, the performance of most of these tools has been tested using simulated insertions, which do not accurately reproduce the complexity of natural insertions., [Results] We have overcome this limitation by building a dataset of insertions from the comparison of two high-quality rice genomes, followed by extensive manual curation. This dataset contains validated insertions of two very different types of TEs, LTR-retrotransposons and MITEs. Using this dataset, we have benchmarked the sensitivity and precision of 12 commonly used tools, and our results suggest that in general their sensitivity was previously overestimated when using simulated data. Our results also show that, increasing coverage leads to a better sensitivity but with a cost in precision. Moreover, we found important differences in tool performance, with some tools performing better on a specific type of TEs. We have also used two sets of experimentally validated insertions in Drosophila and humans and show that this trend is maintained in genomes of different size and complexity., [Conclusions] We discuss the possible choice of tools depending on the goals of the study and show that the appropriate combination of tools could be an option for most approaches, increasing the sensitivity while maintaining a good precision.