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Allelopathy has been considered as a natural method of weed control. Despite the nature of allelochemical compounds has been studied, little is known about the genetic basis underlying allelopathy. However, it is known that rice exhibits diverse allelopathic potentials across varieties, and breeding for rice plants exhibiting allelopathic potential conferring an advantage against weeds in paddy fields would be highly desirable. Knowledge of the gene factors and the identification of the genomic regions responsible for allelopathy would facilitate breeding programs. Taking advantage of the existing genetic diversity in rice, particularly in temperate japonica rice, we conducted a comprehensive investigation into the genetic determinants that contribute to rice allelopathy. Employing Genome-Wide Association Study, we identified four Quantitative Trait Loci, with the most promising loci situated on chromosome 2 and 5. Subsequent inspection of the genes located within these QTLs revealed genes associated with the biosynthesis of secondary metabolites such as Phenylalanine Ammonia Lyase (PAL), a key enzyme in the synthesis of phenolic compounds, and two genes coding for R2R3-type MYB transcription factors. The identification of these two QTLs associated to allelopathy in rice provides a useful tool for further exploration and targeted breeding strategies.

The relationship of transposable elements (TEs) with their host genomes has usually been seen as an arms race between TEs and their host genomes. Consequently, TEs are supposed to amplify by bursts of transposition, when the TE escapes host surveillance, followed by long periods of TE quiescence and efficient host control. Recent data obtained from an increasing number of assembled plant genomes and resequencing population datasets show that TE dynamics is more complex and varies among TE families and their host genomes. This variation ranges from large genomes that accommodate large TE populations to genomes that are very active in TE elimination, and from inconspicuous elements with very low activity to elements with high transposition and elimination rates. The dynamics of each TE family results from a long history of interaction with the host in a genome populated by many other TE families, very much like an evolving ecosystem.

Transposable elements (TEs) are an important source of genome variability. Here, we analyze their contribution to gene expression variability in rice by performing a TE insertion polymorphism expression quantitative trait locus mapping using expression data from 208 varieties from the Oryza sativa ssp. indica and O. sativa ssp. japonica subspecies. Our data show that TE insertions are associated with changes of expression of many genes known to be targets of rice domestication and breeding. An important fraction of these insertions were already present in the rice wild ancestors, and have been differentially selected in indica and japonica rice populations. Taken together, our results show that small changes of expression in signal transduction genes induced by TE insertions accompany the domestication and adaptation of rice populations.

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3ʹ extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.

We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated 238 Mb almond genome size, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27,969 protein-coding genes and 6,747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (P. persica) diverged around 5.88 Mya. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions/kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). TEs have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. TEs may also be at the origin of important phenotypic differences between both species, and in particular, for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.

We analysed the DNA methylation and transcription levels of transposable elements and genes in leaves of Prunus persica and Prunus dulcis and in their F1 hybrid using high-throughput sequencing tecnhologies. We can conclude that the merging of the two parental genomes in the P. persica x P. dulcis hybrid does not result in a “genomic shock” with significant changes in the DNA methylation or in the transcription.

[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.

Transposon insertion polymorphisms can improve prediction of complex agronomic traits in rice compared to using SNPs only, especially when accessions to be predicted are less related to the training set. Abstract: Transposon insertion polymorphisms (TIPs) are significant sources of genetic variation. Previous work has shown that TIPs can improve detection of causative loci on agronomic traits in rice. Here, we quantify the fraction of variance explained by single nucleotide polymorphisms (SNPs) compared to TIPs, and we explore whether TIPs can improve prediction of traits when compared to using only SNPs. We used eleven traits of agronomic relevance from by five different rice population groups (Aus, Indica, Aromatic, Japonica, and Admixed), 738 accessions in total. We assess prediction by applying data split validation in two scenarios. In the within-population scenario, we predicted performance of improved Indica varieties using the rest of Indica accessions. In the across population scenario, we predicted all Aromatic and Admixed accessions using the rest of populations. In each scenario, Bayes C and a Bayesian reproducible kernel Hilbert space regression were compared. We find that TIPs can explain an important fraction of total genetic variance and that they also improve genomic prediction. In the across population prediction scenario, TIPs outperformed SNPs in nine out of the eleven traits analyzed. In some traits like leaf senescence or grain width, using TIPs increased predictive correlation by 30–50%. Our results evidence, for the first time, that TIPs genotyping can improve prediction on complex agronomic traits in rice, especially when accessions to be predicted are less related to training accessions.

Hybridization has been widely used in breeding of cultivated species showing low genetic variability, such as peach (Prunus persica). The merging of two different genomes in a hybrid often triggers a so-called "genomic shock"with changes in DNA methylation and in the induction of transposable element expression and mobilization. Here, we analysed the DNA methylation and transcription levels of transposable elements and genes in leaves of Prunus persica and Prunus dulcis and in an F1 hybrid using high-throughput sequencing technologies. Contrary to the "genomic shock"expectations, we found that the overall levels of DNA methylation in the transposable elements in the hybrid are not significantly altered compared with those of the parental genomes. We also observed that the levels of transcription of the transposable elements in the hybrid are in most cases intermediate as compared with that of the parental species and we have not detected cases of higher transcription in the hybrid. We also found that the proportion of genes whose expression is altered in the hybrid compared with the parental species is low. The expression of genes potentially involved in the regulation of the activity of the transposable elements is not altered. We can conclude that the merging of the two parental genomes in this Prunus persica x Prunus dulcis hybrid does not result in a "genomic shock"with significant changes in the DNA methylation or in the transcription. The absence of major changes may facilitate using interspecific peach x almond crosses for peach improvement.

Transposon Insertion Polymorphisms (TIPs) are a significant source of genetic variation. Previous work (Castanera et al., 2021) has shown that TIPs can improve detection of causative loci on agronomic traits in rice. Here, we quantify the fraction of variance explained by Single Nucleotide Polymorphisms (SNPs) compared to TIPs, and we explore whether TIPs can improve prediction of phenotypes when compared to using only SNPs. We used eleven traits of agronomic relevance from by five different rice population groups (Aus, Indica, Aromatic, Japonica and Admixed), 738 varieties in total. We assess prediction by applying data split validation in two scenarios. In the within population scenario, we predicted performance of improved Indica varieties using the rest of Indica and additional samples. In the across population scenario, we predicted all Aromatic and Admixed samples using the rest of populations. In each scenario, Bayes C and a Bayesian reproducible kernel Hilbert space regression were compared. We find that TIPs can explain an important fraction of total genetic variance, often more than the fraction explained by SNPs, and that they also improve genomic prediction, especially in the across population prediction scenario, where TIPs outperformed SNPs in nine out of the eleven traits analyzed. In some phenotypes like leaf senescence or grain width, using TIPs increased predictive correlation by 40%. Our results evidence, for the first time, that TIPs genotyping can improve prediction on complex agronomic traits in rice, especially when samples to be predicted are less related to training samples.

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.