Biology subjects

Llorente, B.

Publications and source records attributed to Llorente, B..

16 recordsLinked to original sources

bioRxiv2026

A reproductive hierarchy controls mating fates among sibling yeast gametes

Mating strategies shape inbreeding, gene flow and genetic diversity, yet their outcomes remain challenging to quantify. In Saccharomyces, population-genomic models predict mating predominantly between sibling meiotic products, with rare selfing and outcrossing. Here we developed an integrated framework combining multicolor flow cytometry, time-resolved imaging and mathematical modelling to directly quantify mating across scales. Bulk measurements revealed coexisting strategies whose frequencies vary with density, genotype and environment. A minimal stochastic model uses these population-level patterns to infer probabilistic hierarchies among mating modes, which single-tetrad analyses independently support. These reveal divergent and interdependent reproductive trajectories among sibling gametes, with one pair typically mating first and reshaping opportunities for the others. Temporal heterogeneity in germination and mating further diversifies reproductive outcomes and shapes compatibility, as experimental synchronization overcomes temporal reproductive barriers, enabling hybridization. Together, reproductive strategy emerges from heterogeneous gamete fates, raising the possibility that sibling diversification constitutes reproductive bet-hedging.

genetics↗

bioRxiv2026

GenDiMug: a generic inclusive multigraph framework to analyze genomic organizations and -omics data at multiple scales

After decades of investigation, analyses of genome organization remain challenging. This is because genome descriptions are very information-rich and usually rendered as a linear map of genomic features corresponding to an ordered list of position intervals on a nucleic acid macromolecular backbone. This formalism, while accurate, is not fully optimized to mathematically analyze all genome structural properties, in particular the relations between genomic features, nor to integrate large amounts of -omics data, such as transcriptomes, epigenomes, proteomes, meltomes or chromatin accessibility data, which are increasingly available. We therefore developed GenDiMug, a tool that uses a network representation of the genome as a backbone, providing a natural framework to integrate spatial and ontogenetic relationships between genomic features and multi-omics data. We illustrate how this generic approach can be applied to all sorts of genomes, both to visualize genomic topology at contig and chromosomal scales at base-pair resolution, and to analyze genome positional effects using various graph metrics. Using GenDiMug, we further reveal clumping of genes with similar expression levels in eukaryotic genomes and their possible circadian regulation in a photosynthetic alga.

bioinformatics↗

bioRxiv2026

A step toward synthetic endosymbiosis: introducing Escherichia coli and the trypanosomatid endosymbiont Candidatus Kinetoplastibacterium crithidii into mammalian cells using a polyethylene glycol-based fusion protocol

Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring {beta}-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei, we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.

synthetic biology↗

bioRxiv2026

A stepwise route to polyploidy in yeast

Polyploidy, the presence of multiple chromosome sets within a genome, is widespread and has played a major role in genome evolution in plants, fungi, and animals. Although polyploidization is generally attributed to mitotic genome doubling or unreduced gamete fusion, the mechanisms underlying ploidy increases, particularly triploid formation, remain unclear. Here, we identify a novel route to autopolyploidization in Saccharomyces cerevisiae, termed Sporulate-Endoreplicate-Mate (SEM), in which one gamete, or spore, undergoes postmeiotic endoreplication and subsequently mates with a nearby non-endoreplicated sibling spore after ascus breakdown, increasing ploidy by one haploid genome. We experimentally demonstrate stepwise transitions from diploidy to triploidy and from triploidy to tetraploidy. Intact asci can spontaneously generate new triploid and tetraploid strains upon one or two successive SEM cycles. Natural polyploids exhibit genomic signatures consistent with SEM being a major natural route to polyploidization in yeast. SEM establishes triploids as central intermediates in yeast polyploidization, analogous to the triploid bridge described in plants, and raises the possibility that a similar mechanism may contribute to polyploid genome formation in other eukaryotes.

genomics↗

bioRxiv2026

Creating bacterial genomic diversity through large-scale reconfigurations reveals phenotype robustness to organizational genome change

The ability to generate genomic diversity expands opportunities for understanding and engineering biology. Here, we demonstrate on-demand generation of diversity in bacterial genome configurations and its application to probing physiology under altered genome organization. We engineered the fast-growing bacterium Vibrio natriegens to enable large-scale stochastic duplications, translocations, inversions, and deletions, producing populations with a wide range of genome arrangements. We investigated phenotypic robustness to genome reconfigurations and found that distinct genome organizations can support stable physiology, indicating that bacteria may tolerate chromosomal alterations more readily than previously appreciated. Our work provides a framework for advancing the understanding and engineering of bacterial genomes and suggests that genome reconfigurations may contribute to phenogenetic drift, allowing for evolutionary exploration while preserving phenotype.

synthetic biology↗

bioRxiv2025

Repeated losses of self-fertility shaped heterozygosity and polyploidy in yeast evolution

Evolutionary transitions in mating strategy have profound consequences for genetic variation and adaptation. In Saccharomyces cerevisiae, mating-type switching is a central feature of the life cycle that enables homothallism, i.e., mating between mitotic descendants of the same haploid cell. Yet heterothallic isolates that have lost this ability are found across diverse niches, indicating that this trait is polymorphic. Here we experimentally characterized loss of mating-type switching in a representative panel of strains. Analysis of 117 telomere- to-telomere genome assemblies revealed multiple independent loss-of-function mutations in the Ho endonuclease gene and structural variants in the silent HML and HMR cassettes, the three loci essential for switching. We estimated that at least 13 independent transitions from homothallism to heterothallism have occurred in the species history. Analysis of the HO genotype of 2,915 strains show that at least 27% are heterothallic. We found that heterothallism is strongly associated with polyploidy and elevated genome-wide heterozygosity, although the strength of these associations varies between populations. Heterothallic isolates are most prevalent in domesticated and clinical clades, consistent with an origin linked to human-associated environments. However, they are also found, though less frequently, in natural niches. Signatures of recombination in HO sequences suggest that outcrossing contributed to the ecological and geographical distribution of the trait. Our findings reveal that mating-type switching has undergone repeated losses in S. cerevisiae evolution, with major consequences for genome architecture and ecological diversification. Significance StatementMating-type switching evolved multiple times in yeasts, enabling haploid selfing, which became a key feature of their life cycles. We show that this trait has been lost repeatedly in the history of Saccharomyces cerevisiae, producing heterothallic isolates that cannot switch mating type. Analysis of thousands of genomes reveals that these losses involve multiple mutations in the Ho endonuclease and structural changes in the silent mating-type cassettes. Heterothallism is associated with polyploidy and increased genome-wide heterozygosity. It is more frequent in domesticated and clinical lineages, but also occurs in natural ecological niches. Our findings illustrate how repeated changes in a single life-history trait can reshape genome architecture and highlight the dynamic interplay between genetics, environment, and evolution in a model eukaryote.

evolutionary biology↗

bioRxiv2023

Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments

The relevance of plants as food is expected to grow for a more sustainable diet. In this new context, improving the nutritional quality of plant-derived foods is a must. Biofortification of green leafy vegetables with pro-vitamin A carotenoids such as {beta}-carotene has remained challenging to date. Here we combined two strategies to achieve this goal. One of them (that we call strategy C) involves producing {beta}-carotene in extraplastidial locations of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach (that we refer to as strategy P) involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in some leaves, leaving other non-engineered leaves to sustain normal plant growth. Combination of these two strategies resulted in a 5- fold increase in the amount of {beta}-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve {beta}-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with high-light treatments not only improved {beta}-carotene accumulation but it also resulted in a much higher bioaccessibility. Combination of strategies C and P together with a high-light treatment increased the levels of accessible {beta}-carotene 30-fold compared to controls. We further demonstrate that stimulating plastoglobule proliferation with strategy P but also with high-light alone can also stimulate and hence improve {beta}-carotene contents and bioaccessibility in edible lettuce (Lactuca sativa) leaves, unveiling the power of non-GMO approaches for leaf biofortification.

plant biology↗

bioRxiv2022

Remarkably high rate of meiotic recombination in the fission yeast Schizosaccharomyces pombe

In most eukaryotes, the number of meiotic crossovers (COs) is limited to 1-3 per chromosome, which are prevented from occurring close to one another by CO interference. The fission yeast Schizosaccharomyces pombe, an exception to this general rule, lacks CO interference and seems to have the highest CO number per chromosome. However, global CO frequency was indirectly estimated in this species, raising doubts about this exceptional recombination level. Here, we used an innovative strategy to directly determine COs genome-wide in S. pombe. We confirm the absence of crossover interference and reveal the presence of co-variation in CO number across chromosomes within tetrads, suggesting that a limiting pro-CO factor varies stochastically between meiocytes. CO number per chromosome varies linearly with chromosome size, with the three chromosomes having, on average, 15.9, 12.5, and 7.0 COs, respectively. This is significantly lower than previous estimates but reinforces S. pombes exceptional status as the eukaryote with the highest CO number per chromosome described to date and among the species with the highest rate of COs per unit of DNA.

genetics↗

bioRxiv2022

Laboratory evolution and polyploid SCRaMbLE reveal genomic plasticity to synthetic chromosome defects and rearrangements

We have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron (NOG2) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

synthetic biology↗

bioRxiv2022

Comprehensive analysis of cis- and trans-acting factors affecting Break-Induced Replication

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating BIR efficiency, we found that BIR efficiency is the highest close to chromosome ends. The variations of BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of BIR depends on the length of DNA already synthesized. BIR is more susceptible to disruption during the synthesis of the first [~]35-40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.

genetics↗

bioRxiv2022

RecombineX: a generalized computational framework for automatic high-throughput gamete genotyping and tetrad-based recombination analysis

Meiotic recombination is an essential biological process that ensures faithful chromosome segregation and promotes parental allele shuffling. Tetrad analysis is a powerful approach to quantify the genetic makeups and recombination landscapes of meiotic products. Here we present RecombineX (https://github.com/yjx1217/RecombineX), a generalized computational framework that automates the full workflow of marker identification, gamete genotyping, and tetrad-based recombination profiling based on any organisms and genetic backgrounds with batch processing capability. Aside from conventional reference-based analysis, RecombineX can also perform analysis based on parental genome assemblies, which enables analyzing meiotic recombination landscapes in their native genomic contexts. Additional features such as copy number variation profiling and missing genotype inference further enhance downstream analysis. RecombineX also includes a dedicate module for simulating the genomes and reads of recombinant tetrads, which enables fine-tuned simulation-based hypothesis testing. This simulation module revealed the power and accuracy of RecombineX even when analyzing tetrads with very low sequencing depths (e.g., 1-2X). Tetrad sequencing data from the budding yeast Saccharomyces cerevisiae and green alga Chlamydomonas reinhardtii were further used to demonstrate the accuracy and robustness of RecombineX for organisms with both small and large genomes, manifesting RecombineX as an all-around one stop solution for future tetrad analysis. Author SummaryMeiosis is a fundamental cellular process that ensures faithful chromosome segregation and promotes allele shuffling. Tetrad analysis, which isolates and genotypes all four meiotic products (i.e., tetrad) derived from a single meiosis, remains the most straightforward and powerful way of studying meiotic recombination and its modulators at fine scales. The wide application of tetrad analysis in yeasts, filamentous fungi, green algae, and land plants have substantially expand our understanding of meiotic recombination in terms of both genome-wide landscapes and molecular mechanisms. Here we described the first generalized computational framework named RecombineX that automates the full workflow of tetrad analysis based on any organisms and genetic backgrounds. In addition, aside from conventional reference-based analysis, RecombineX can also perform analysis based on parental genome assemblies, which enables analyzing meiotic recombination landscapes in their native genomic contexts. Using both simulated and real tetradsequencing data, we further demonstrated RecombineXs trustable performance, versatile usage, and batch-processing capability, manifesting RecombineX as an all-around one stop solution for tetrad analysis. Especially considering that meiotic gamete genome sequencing from different natural and mutant backgrounds can now be acquired, we expect RecombineX to become a popular tool that empowers future tetrad analysis across different genetic backgrounds and species.

bioinformatics↗

bioRxiv2021

Evolution of quantitative trait locus hotspots in yeast species

Dissecting the genetic basis of complex trait remains a real challenge. The budding yeast Saccharomyces cerevisiae has become a model organism for studying quantitative traits, successfully increasing our knowledge in many aspects. However, the exploration of the genotype-phenotype relationship in non-model yeast species could provide a deeper insight into the genetic basis of complex traits. Here, we have studied this relationship in the Lachancea waltii species which diverged from the S. cerevisiae lineage prior to the whole-genome duplication. By performing linkage mapping analyses in this species, we identified 86 quantitative trait loci (QTL) affecting growth fitness in a large number of conditions. The distribution of these loci across the genome has revealed two major QTL hotspots. A first hotspot corresponds to a general fitness QTL, impacting a wide range of conditions. By contrast, the second hotspot highlighted a fitness trade-off with a disadvantageous allele for drug-free conditions which proved to be advantageous in the presence of several drugs. Finally, the comparison of the detected QTL in L. waltii with those which had been previously identified for the same traits in a closely related species, Lachancea kluyveri, clearly revealed the absence of interspecific conservation of these loci. Altogether, our results expand our knowledge on the variation of the QTL landscape across different yeast species.

genetics↗

bioRxiv2018

Separable roles of the DNA damage response kinase Mec1(ATR) and its activator Rad24(RAD17) within the regulation of meiotic recombination

During meiosis, programmed DNA double-strand breaks (DSBs) are formed by the topoisomerase-like enzyme, Spo11, activating the DNA damage response (DDR) kinase Mec1ATR via the checkpoint clamp loader, Rad24RAD17. At single loci, loss of Mec1 and Rad24 activity alters Spo11-dependent DSB formation and recombination outcome, but their genome-wide roles have not been examined in detail. Here we utilise two distinct methods to characterise the roles Mec1 and Rad24 play in meiotic recombination--deletion of the mismatch repair protein, Msh2, and control of meiotic prophase length via regulation of the Ndt80 transcription factor--to enable genome-wide mapping of meiotic progeny. In line with previous studies, we observe a reduction in the frequency of recombination upon deletion of RAD24--driven by a shortened prophase. By contrast, loss of Mec1 function increases recombination frequency, consistent with its role in DSB trans-interference. Despite this difference in recombination rate, complex, multi-chromatid events initiated by closely spaced DSBs--a rare event in wild type cells--occur more frequently in the absence of either Rad24 or Mec1, suggesting a loss of spatial regulation at the level of DSB formation. We further demonstrate that Mec1 and Rad24 also have important, yet distinct, roles in the spatial regulation of crossovers (COs). Specifically, excess DSBs forming in the absence of Mec1 are disproportionately channelled into the non-crossover (NCO) and non-interfering CO pathways, reducing the global strength of interference without an explicit change in the frequency of interfering COs. In direct contrast, loss of Rad24 weakens interference via a reduction in the apparent number of interfering COs--similar, but less extreme, to the phenotype of ZMM mutants such as zip3{Delta}. Collectively, our results highlight novel and unique roles for Rad24 within meiotic recombination--beyond those mediated by activation of Mec1--and describe new roles for the DDR in several important aspects of meiosis.

genetics↗

bioRxiv2018

Mismatch repair impedes meiotic crossover interference

Sequence divergence, mediated by the anti-recombinogenic activity of mismatch repair (MMR), forms a barrier to meiotic recombination and in turn the formation of viable gametes. However, rather than MMR acting as a non-specific impediment to meiotic recombination, here we provide evidence that at regions of greater sequence divergence MMR preferentially suppresses interfering (class I) crossovers (COs). Specifically, as measured in two Saccharomyces cerevisiae hybrids containing thousands of DNA-sequence polymorphisms, removal of MMR components increases both the frequency of CO formation and the uniformity of the observed CO distribution. At fine scale, CO positions are skewed away from polymorphic regions in MMR-proficient cells, but, critically, not when members of the class I CO pathway, MSH4 or ZIP3, are inactivated. These findings suggest that class I COs are more sensitive to heteroduplex DNA arising during recombination. Simulations and analysis of Zip3 foci on meiotic chromosomes support roles for Msh2 both early and late in the class I CO maturation process. Collectively, our observations highlight an unexpected interaction between DNA sequence divergence, MMR, and meiotic class I CO control, thereby intimately linking the regulation of CO numbers and their distribution to pathways contributing to reproductive isolation and eventual speciation.

genetics↗

bioRxiv2017

Mechanistic view and genetic control of DNA recombination during meiosis

Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here we revisit these models by analyzing meiotic heteroduplex DNA tracts genome-wide in combination with meiotic DNA double-strand break (DSB) locations. We provide unprecedented support to the synthesis-dependent strand annealing model and establish estimates of its associated template switching frequency and polymerase processivity. We show that resolution of double Holliday junctions (dHJs) is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions. The suspected dHJ resolvase Mlh1-3 as well as Mlh1-2, Exo1 and Sgs1 promote asymmetric positioning of crossover intermediates relative to the initiating DSB and bidirectional conversions. Finally, we show that crossover-biased dHJ resolution depends on Mlh1-3, Exo1, Msh5 and to a lesser extent on Sgs1. These properties are likely conserved in eukaryotes containing the ZMM proteins, which includes mammals.

genetics↗

bioRxiv2017

Variation Of The Meiotic Recombination Landscape And Properties Over A Broad Evolutionary Distance In Yeasts

Meiotic recombination is a major factor of genome evolution, deeply characterized in only a few model species, notably the yeast Saccharomyces cerevisiae. Consequently, little is known about variations of its properties across species. In this respect, we explored the recombination landscape of Lachancea kluyveri, a protoploid yeast species that diverged from the Saccharomyces genus more than 100 million years ago and we found striking differences with S. cerevisiae. These variations include a lower recombination rate, a higher frequency of chromosomes segregating without any crossover and the absence of recombination on the chromosome arm containing the sex locus. In addition, although well conserved within the Saccharomyces clade, the S. cerevisiae recombination hotspots are not conserved over a broader evolutionary distance. Finally and strikingly, we found evidence of frequent reversion of meiotic commitment to mitotic growth allowing allele shuffling without meiosis completion. Identification of this major but underestimated evolutionary phenomenon illustrates the relevance of exploring non-model species.\n\nAuthor summaryMeiotic recombination promotes accurate chromosome segregation and genetic diversity. To date, the mechanisms and rules lying behind recombination were dissected using model organisms such as the budding yeast Saccharomyces cerevisiae. To assess the conservation and variation of this process over a broad evolutionary distance, we explored the meiotic recombination landscape in Lachancea kluyveri, a budding yeast species that diverged from S. cerevisiae more than 100 million years ago. The meiotic recombination map we generated revealed that the meiotic recombination landscape and properties significantly vary across distantly related yeast species, supporting that recombination hotspots conservation across yeast species is likely associated to the conservation of synteny. Finally, the frequent meiotic reversions we observed led us to re-evaluate their evolutionary importance.

genomics↗