Biology subjects

Llauro, C.

Publications and source records attributed to Llauro, C..

8 recordsLinked to original sources

bioRxiv2026

Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis

RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.

plant biology↗

bioRxiv2026

A cryptic hybrid zone reveals the genomic basis of flower colour variation in a plant with a large and complex genome

Flower colour is known to be a classic magic trait, undergoing divergent selection pressures exerted by distinct pollinators and involved in reproductive isolation. However, variation at this trait may also results from other eco-evolutionary factors whose interpretation requires throughout context-specific analyses. Here, we investigated the eco-evolutionary causes of pink- and yellow-flowered morphs in Pedicularis comosa where their parapatric ranges form a contact zone in the East of the Pyrenees Mountains. First, we generated a near-chromosome-scale reference genome assembly for this species. We then used a Genotyping By Sequencing approach to infer patterns of genetic diversity and differentiation between populations and morphs at a total of 158 individuals from 11 localities. We found that neutral genetic structure is primarily consistent with geography rather than with colour. However, admixture analyses and clines suggest the existence of a cryptic hybrid zone between morphs. Outlier detection methods and examination of locus-by-locus cline features, then allowed to pinpoint candidate loci to explain colour variation. To gain insight into the functional aspects of these loci, we finally analysed floral transcriptomes and quantified pigments using Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and confirmed the involvement of key genes in the anthocyanin metabolic pathway (e.g. DFR, FLS) and associated pigments (e.g. cyanidin and delphinidin). Our results show that implementing a highly integrative multi-omic approach can allow unraveling the genetic basis and the eco-evolutionary significance of adaptive traits with even very limited previous knowledge, on species with large and complex genomes.

evolutionary biology↗

bioRxiv2026

Phylotranscriptomics Allows Distinguishing Major Gene Flow Events from Incomplete Lineage Sorting in Rapidly Diversifying Mimetic Orchids (Genus Ophrys)

Ophrys orchids (or bee orchids) provide an outstanding example of a plant adaptive radiation. Over the last five million years, this genus has diversified into hundreds of taxa as a result of its unconventional pollination strategy, known as sexual swindling. However, the rapid and substantial diversification of this genus, combined with its capacity for hybridisation and large genome size, poses significant challenges in addressing its systematics. We used phylotranscriptomics as a genome complexity reduction technique to infer the phylogenetic relationships among Ophrys main lineages. More than seven thousand gene trees enabled us to determine the relative contributions of gene flow and incomplete lineage sorting (ILS) in Ophrys evolution. First, we propose a new phylogenetic hypothesis for the genus with an unprecedented resolution that largely confirms the relationships between the main Ophrys lineages, but also provides new insights within each sub-genera. By combining phylogenetic network inference with introgression analyses based on gene tree topologies and branch lengths, we then show that the numerous phylogenetic incongruences among gene tree topologies result from a pervasive background of ILS, over which stand out several well-supported, ancient and potentially adaptive gene flow events between lineages. These major gene flow events provide a new perspective on the evolution of the Ophrys genus and its pollination, questioning previous hypotheses inferred without considering its reticulate evolution, and providing a better understanding of discrepancies observed among previous phylogenetic studies of the genus.

evolutionary biology↗

bioRxiv2025

Role of methylation and siRNA on differential allelic expression in a hybrid of distantly related citrus species.

DNA methylation plays a central role in the regulation of gene expression. In plants, methylation occurs in the CG, CHG and CHH contexts, via distinct DNA methyltransferases including MET1, CMT3 and the RNA-directed DNA Methylation (RdDM) pathway via DRM2. In interspecific hybrids, these epigenetic mechanisms are confronted to a mixed small RNA population and two subgenomes harbouring specific methylation patterns, therefore generating unique expression profiles. The aim of this work was to understand these regulations by analysing gene expression, DNA methylation and small RNAs in a Citrus hybrid resulting from the cross between C. reticulata (mandarin) and C. australasica (finger lime). Haplotype-resolved subgenomes assembly identified hundreds of allele-specifically expressed genes. Asymmetric reprogramming of methylation was observed, in particular an increase in CHH in C. australasica haplotype. Surprisingly, CHH methylation, usually associated with gene silencing, was correlated here with increased expression, but also 24nt small RNA populations at their promoter regions. Similar analyses of the parental lines and other citrus species suggest the correlation between CHH methylation-enriched promoter and high expression level is not due to the hybridization, but seem to be generally true for all citrus. These observations suggest that, in citrus fruit, RdDM could activate transcription. This work also provides a full pipeline to analyse the expression profiles and DNA methylation in complex hybrids, which could be crucial for anticipating varieties resistant to diseases and the current threats affecting citriculture such as the Huanglongbing disease.

plant biology↗

bioRxiv2025

Non-invasive eDNA reveals the ecological and genetic status of the Western Capercaillie (Tetrao urogallus aquitanicus) in the Eastern Pyrenees

The Anthropocene era is expected to bring about significant biodiversity and habitat loss for many species. These geographical changes, whether driven by climatic or anthropogenic factors, are likely to lead to considerable alterations in population size, structure, and genetic diversity. Monitoring natural populations is therefore essential to assess these impacts and enable informed conservation strategies for threatened species. The Western Capercaillie (Tetrao urogallus, L. 1758) has a widespread distribution in Boreal forests but fragmented in mountainous regions of the Palearctic, and is locally threatened by climate change, habitat destruction, and human disturbance. Our study focused on the eastern population of the subspecies T. u. aquitanicus, which is endemic to the Pyrenees mountains. The monitoring of this population has relied on direct methods and no genetic information had been generated so far. Here, we conducted a molecular study based on 229 non-invasive samples (faeces) to assess the ecological and genetic status of local population in the Catalan Nature Reserves of the Pyrenees-Orientales (Occitanie region, France). At the individual level, we assessed multi-locus genotypes, sexing, levels of inbreeding, stress level (Fecal Corticosterone Metabolites; FCMs) and diet. At the population level, we assessed sex ratio, genetic diversity and structure. We identified 62 individuals with a balanced sex ratio and estimated a census size of 79 individuals [95%CI = 68-92] in the study area. Genetic diversity was low and suggested significant inbreeding levels. FCM levels were lower in birds of areas considered as disturbed by humans and metabarcoding approach indicated a geographical structuring of diet composition at the reserve scale, with individuals exhibiting feeding behavior upon only one or few plant species. Our estimate of population census size was higher with figures assessed from lek counts, and the genetic approach provided additional insights on this population, establishing a baseline that will support conservation management plans.

ecology↗

bioRxiv2021

Characterization Of Interspecific Gene Flows at The Genome-wide Level in a Natural Ecosystem The Massane Forest Reveals New Insights Into Horizontal Transfer In plants.

BackgroundHorizontal transfer (HT) refers to the exchange of genetic material between divergent species by mechanisms other than reproduction. In recent years, several studies have demonstrated HTs in eukaryotes, particularly in the context of parasitic relationships and in model species. However, very little is known about HT in natural ecosystems, especially those involving non-parasitic wild species, and the nature of the ecological relationships that promote these HTs. ResultsIn this work, we conducted a pilot study investigating HTs by sequencing the genomes of 17 wild non-model species from a natural ecosystem, the Massane forest, located in southern France. To this end, we developed a new computational pipeline called INTERCHANGE that is able to characterize HTs at the whole genome level without prior annotation and directly in the raw sequencing reads. Using this pipeline, we identified 12 HT events, half of which occurred between lianas and trees. We found that mainly low copy number LTRs retrotransposons from the Copia superfamily were transferred between these wild plant species, especially those of the Ivan and Ale lineages. ConclusionThis study revealed a possible new route for HTs between non-parasitic plants and provides new insights into the genomic characteristics of horizontally transferred DNA in plant genomes.

genomics↗

bioRxiv2020

Whole plastid genome-based phylogenomics supports an inner placement of the O. insectifera group rather than a basal position in the rapidly diversifying Ophrys genus (Orchidaceae)

Some lineages of the Orchid genus Ophrys exhibit among the highest diversification rates reported so far. As a consequence of a such intense and rapid evolution, the systematics and the taxonomy of this genus remains unclear. A hybrid assembly approach based-on long- and short-read genomic data allowed us to outperform classical methods to successfully assemble whole plastid genomes for two new Ophrys species: O. aymoninii and O. lutea. Along with three other previously Ophrys plastid genome sequences, we then reconstructed the first whole plastome-based molecular phylogeny including representatives of the three mains recognized Ophrys lineages. Our results support the placement of the O. insectifera clade as sister group of non-basal Ophrys rather than a basal position. Our findings corroborate recent results obtained from genomic data (RAD-seq and transcriptomes) but contrast with previous ones. These results therefore confirm that molecular phylogenetic hypotheses based on a limited number of loci (e.g. nrITS, matK, rbcL) may have provided a biased picture of phylogenetic relationships within Ophrys and possibly other plant taxa.

evolutionary biology↗

bioRxiv2020

Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response

Among the hundreds of ribosomal RNA (rRNA) gene copies organized as tandem repeats in the nucleolus organizer regions (NORs), only a portion is usually actively expressed in the nucleolus and participate in the ribosome biogenesis process. The role of these extra-copies remains elusive, but previous studies suggested their importance in genome stability and global gene expression. Because the nucleolus is also a platform for nuclear organization, we tested the impact of a decreased amount of rRNA gene copies on the Arabidopsis thaliana 3D genome organization and stability, using an A. thaliana line only containing 20% of rRNA gene copies (20rDNA line). Compared to the wild-type Col-0, the 20rDNA line shows several signs of genomic instability, such as variations in 3D genome organization, spontaneous double-strand breaks accumulation, transcriptomic changes, and higher DNA methylation level. Strikingly, using genomic and microscopic approaches, we identified seven large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. As a consequence, more than 600 genes were duplicated, often associated with an increase in their expression level. Among them, we found several upregulated genes involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and we discuss their potential implications on plant genome evolution.

plant biology↗