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PREMISE: In plants, whole-genome duplication (WGD) is a common mutation with profound evolutionary potential. Given the costs associated with a superfluous genome copy, polyploid establishment is enigmatic. However, in the right environment, immediate phenotypic changes following WGD can facilitate establishment. Metabolite abundances are the direct output of the cell's regulatory network and determine much of the impact of environmental and genetic change on the phenotype. While it is well known that an increase in the bulk amount of genetic material can increase cell size, the impact of gene dosage multiplication on the metabolome remains largely unknown. METHODS: We used untargeted metabolomics on four genetically distinct diploid-neoautotetraploid pairs of the greater duckweed, Spirodela polyrhiza, to investigate how WGD affects metabolite abundances per cell and per biomass. RESULTS: Autopolyploidy increased metabolite levels per cell, but the response of individual metabolites varied considerably. However, the impact on metabolite level per biomass was restricted because the increased cell size reduced the metabolite concentration per cell. Nevertheless, we detected both quantitative and qualitative effects of WGD on the metabolome. Many effects were strain-specific, but some were shared by all four strains. CONCLUSIONS: The nature and impact of metabolic changes after WGD depended strongly on the genotype. Dosage effects have the potential to alter the plant metabolome qualitatively and quantitatively, but were largely balanced out by the reduction in metabolite concentration due to an increase in cell size in this species.
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PREMISE: Whole-genome duplication (WGD, polyploidization) has been identified as a driver of genetic and phenotypic novelty, having pervasive consequences for the evolution of lineages. While polyploids are widespread, especially among plants, the long-term establishment of polyploids is exceedingly rare. Genome doubling commonly results in increased cell sizes and metabolic expenses, which may be sufficient to modulate polyploid establishment in environments where their diploid ancestors thrive. METHODS: We developed a mechanistic simulation model of photosynthetic individuals to test whether changes in size and metabolic efficiency allow autopolyploids to coexist with, or even invade, ancestral diploid populations. Central to the model is metabolic efficiency, which determines how energy obtained from size-dependent photosynthetic production is allocated to basal metabolism as opposed to somatic and reproductive growth. We expected neopolyploids to establish successfully if they have equal or higher metabolic efficiency as diploids or to adapt their life history to offset metabolic inefficiency. RESULTS: Polyploid invasion was observed across a wide range of metabolic efficiency differences between polyploids and diploids. Polyploids became established in diploid populations even when they had a lower metabolic efficiency, which was facilitated by recurrent formation. Competition for nutrients is a major driver of population dynamics in this model. Perenniality did not qualitatively affect the relative metabolic efficiency from which tetraploids tended to establish. CONCLUSIONS: Feedback between size-dependent metabolism and energy allocation generated size and age differences between plants with different ploidies. We demonstrated that even small changes in metabolic efficiency are sufficient for the establishment of polyploids.
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Following whole-genome duplication (WGD), duplicate gene pairs (homoeologs) can evolve varying degrees of expression divergence. However, the determinants influencing these relative expression level differences (RFPKM) between homoeologs remain elusive. Here, we analyzed the RFPKM between homoeologs in three angiosperms, Nymphaea colorata, Nelumbo nucifera, and Acorus tatarinowii, all having undergone a single WGD since the origin of angiosperms. Our results show significant positive correlations in RFPKM of homoeologs among tissues within the same species, and among orthologs across these three species, indicating convergent expression balance/bias between homoeologous gene copies following independent WGDs. We linked RFPKM between homoeologs to gene attributes associated with dosage balance constraints, such as protein-protein interactions, lethal-phenotype scores in Arabidopsis (Arabidopsis thaliana) orthologs, domain numbers, and expression breadth. Notably, homoeologs with lower RFPKM often had more interactions and higher lethal-phenotype scores, indicating selective pressures favoring balanced expression. Also, homoeologs with lower RFPKM were more likely to be retained after WGDs in angiosperms. Within Nelumbo, greater RFPKM between homoeologs correlated with increased cis- and trans-regulatory differentiation between species, highlighting the ongoing escalation of gene expression divergence. We further found that expression degeneration in one copy of homoeologs is inclined towards nonfunctionalization. Our research highlights the importance of balanced expression, shaped by dosage balance constraints, in the evolutionary retention of homoeologs in plants.
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Hybridization blurs species boundaries and leads to intertwined lineages resulting in reticulate evolution. Polyploidy, the outcome of whole genome duplication (WGD), has more recently been implicated in promoting and facilitating hybridization between polyploid species, potentially leading to adaptive introgression. However, because polyploid lineages are usually ephemeral states in the evolutionary history of life it is unclear whether WGD-potentiated hybridization has any appreciable effect on their diploid counterparts. Here, we develop a model of cytotype dynamics within mixed-ploidy populations to demonstrate that polyploidy can in fact serve as a bridge for gene flow between diploid lineages, where introgression is fully or partially hampered by the species barrier. Polyploid bridges emerge in the presence of triploid organisms, which despite critically low levels of fitness, can still allow the transfer of alleles between diploid states of independently evolving mixed-ploidy species. Notably, while marked genetic divergence prevents polyploid-mediated interspecific gene flow, we show that increased recombination rates can offset these evolutionary constraints, allowing a more efficient sorting of alleles at higher-ploidy levels before introgression into diploid gene pools. Additionally, we derive an analytical approximation for the rate of gene flow at the tetraploid level necessary to supersede introgression between diploids with nonzero introgression rates, which is especially relevant for plant species complexes, where interspecific gene flow is ubiquitous. Altogether, our results illustrate the potential impact of polyploid bridges on the (re)distribution of genetic material across ecological communities during evolution, representing a potential force behind reticulation.
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Flujo Génico , Hibridación Genética , Modelos Genéticos , Poliploidía , Evolución Molecular , Diploidia , AlelosRESUMEN
Hybridization, the process of crossing individuals from diverse genetic backgrounds, plays a pivotal role in evolution, biological invasiveness, and crop breeding. At the transcriptional level, hybridization often leads to complex nonadditive effects, presenting challenges for understanding its consequences. Although standard transcriptomic analyses exist to compare hybrids to their progenitors, such analyses have not been implemented in a software package, hindering reproducibility. We introduce hybridexpress, an R/Bioconductor package designed to facilitate the analysis, visualization, and comparison of gene expression patterns in hybrid triplets (hybrids and their progenitors). hybridexpress provides users with a user-friendly and comprehensive workflow that includes all standard comparative analyses steps, including data normalization, calculation of midparent expression values, sample clustering, expression-based gene classification into categories and classes, and overrepresentation analysis for functional terms. We illustrate the utility of hybridexpress through comparative transcriptomic analyses of cotton allopolyploidization and rice root trait heterosis. hybridexpress is designed to streamline comparative transcriptomic studies of hybrid triplets, advancing our understanding of evolutionary dynamics in allopolyploids, and enhancing plant breeding strategies. hybridexpress is freely accessible from Bioconductor (https://bioconductor.org/packages/HybridExpress) and its source code is available on GitHub (https://github.com/almeidasilvaf/HybridExpress).
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Perfilación de la Expresión Génica , Hibridación Genética , Oryza , Programas Informáticos , Transcriptoma , Oryza/genética , Transcriptoma/genética , Gossypium/genética , Vigor Híbrido/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética , PoliploidíaRESUMEN
Lasiodiplodia hormozganensis, initially recognized as a fungal plant pathogen, is recognized now acknowledged as a potential threat to humans. However, our understanding of the pathogenesis mechanisms of Lasiodiplodia species remains limited, and the impact of temperature on its pathogenicity is unclear. This study aims to elucidate the effects of temperature on the biology of L. hormozganensis, focusing on the expression of pathogenesis-related molecules and its ability to function as a cross-kingdom pathogen. We conducted experiments at two different temperatures, 25 and 37 °C, analyzing the proteome and transcriptome of L. hormozganensis. Using strain CBS339.90, initially identified as L. theobromae but confirmed through ITS and tef1-α sequence analysis to be L. hormozganensis, we aimed to understand the fungus's protein expression under varying temperature conditions. Results from the functional analysis of the secretome at 25 °C showed a noteworthy presence of proteins related to carbohydrate metabolism, catabolism, plant cell wall degradation, and pathogenesis. However, when grown at 37 °C, the fungus exhibited an increased production of stress response and pathogenesis-related proteins. Our findings identified various pathways crucial for pathogenesis in both plants and humans, suggesting that L. hormozganensis possesses the genetic foundation to infect both hosts. Specific pathogenesis-related proteins, including the phytotoxin snodprot1, aspartic protease aspergillopepsin, and virulence protein SSD1, were also identified. Concluding, we propose a possible mechanism of how L. hormozganensis adapts to different temperatures. The shift in temperature results in the expression of genes that favor human related pathogenesis molecules.
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Ascomicetos , Temperatura , Ascomicetos/fisiología , Ascomicetos/genética , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , TranscriptomaRESUMEN
MOTIVATION: Major improvements in sequencing technologies and genome sequence assembly have led to a huge increase in the number of available genome sequences. In turn, these genome sequences form an invaluable source for evolutionary, ecological, and comparative studies. One kind of analysis that has become routine is the search for traces of ancient polyploidy, particularly for plant genomes, where whole-genome duplication (WGD) is rampant. RESULTS: Here, we present a major update of a previously developed tool wgd, namely wgd v2, to look for remnants of ancient polyploidy, or WGD. We implemented novel and improved previously developed tools to (a) construct KS age distributions for the whole-paranome (collection of all duplicated genes in a genome), (b) unravel intragenomic and intergenomic collinearity resulting from WGDs, (c) fit mixture models to age distributions of gene duplicates, (d) correct substitution rate variation for phylogenetic placement of WGDs, and (e) date ancient WGDs via phylogenetic dating of WGD-retained gene duplicates. The applicability and feasibility of wgd v2 for the identification and the relative and absolute dating of ancient WGDs is demonstrated using different plant genomes. AVAILABILITY AND IMPLEMENTATION: wgd v2 is open source and available at https://github.com/heche-psb/wgd.
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Duplicación de Gen , Genoma de Planta , Filogenia , Poliploidía , Evolución Molecular , Programas Informáticos , Genómica/métodosRESUMEN
Increasing studies suggest that the biased retention of stress-related transcription factors (TFs) after whole-genome duplications (WGDs) could rewire gene transcriptional networks, facilitating plant adaptation to challenging environments. However, the role of posttranscriptional factors (e.g. RNA-binding proteins, RBPs) following WGDs has been largely ignored. Uncovering thousands of RBPs in 21 representative angiosperm species, we integrate genomic, transcriptomic, regulatomic, and paleotemperature datasets to unravel their evolutionary trajectories and roles in adapting to challenging environments. We reveal functional enrichments of RBP genes in stress responses and identify their convergent retention across diverse angiosperms from independent WGDs, coinciding with global cooling periods. Numerous RBP duplicates derived from WGDs are then identified as cold-induced. A significant overlap of 29 orthogroups between WGD-derived and cold-induced RBP genes across diverse angiosperms highlights a correlation between WGD and cold stress. Notably, we unveil an orthogroup (Glycine-rich RNA-binding Proteins 7/8, GRP7/8) and relevant TF duplicates (CCA1/LHY, RVE4/8, CBF2/4, etc.), co-retained in different angiosperms post-WGDs. Finally, we illustrate their roles in rewiring circadian and cold-regulatory networks at both transcriptional and posttranscriptional levels during global cooling. Altogether, we underline the adaptive evolution of RBPs in angiosperms after WGDs during global cooling, improving our understanding of plants surviving periods of environmental turmoil.
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Magnoliopsida , Magnoliopsida/genética , Filogenia , Evolución Molecular , Genoma de Planta , Duplicación de Gen , Plantas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio high-fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the 2 parents of the well-studied F1 hybrid "84K" (Populus alba × Populus tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from 2 small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the 2 subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive-expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.
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Alelos , Genoma de Planta , Populus , Populus/genética , Genoma de Planta/genética , Regulación de la Expresión Génica de las Plantas , Haplotipos/genética , Hibridación Genética , Aprendizaje AutomáticoRESUMEN
Hormones control developmental and physiological processes, often by regulating the expression of multiple genes simultaneously or sequentially. Crosstalk between hormones and epigenetics is pivotal to dynamically coordinate this process. Hormonal signals can guide the addition and removal of epigenetic marks, steering gene expression. Conversely, DNA methylation, histone modifications and non-coding RNAs can modulate regional chromatin structure and accessibility and regulate the expression of numerous (hormone-related) genes. Here, we provide a review of the interplay between the classical insect hormones, ecdysteroids and juvenile hormones, and epigenetics. We summarize the mode-of-action and roles of these hormones in post-embryonic development, and provide a general overview of epigenetic mechanisms. We then highlight recent advances on the interactions between these hormonal pathways and epigenetics, and their involvement in development. Furthermore, we give an overview of several 'omics techniques employed in the field. Finally, we discuss which questions remain unanswered and possible avenues for future research.
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Metilación de ADN , Epigénesis Genética , Animales , Metilación de ADN/genética , Transducción de Señal , Insectos/genética , HormonasRESUMEN
We present chromosome-level genome assemblies from representative species of three independently evolved seagrass lineages: Posidonia oceanica, Cymodocea nodosa, Thalassia testudinum and Zostera marina. We also include a draft genome of Potamogeton acutifolius, belonging to a freshwater sister lineage to Zosteraceae. All seagrass species share an ancient whole-genome triplication, while additional whole-genome duplications were uncovered for C. nodosa, Z. marina and P. acutifolius. Comparative analysis of selected gene families suggests that the transition from submerged-freshwater to submerged-marine environments mainly involved fine-tuning of multiple processes (such as osmoregulation, salinity, light capture, carbon acquisition and temperature) that all had to happen in parallel, probably explaining why adaptation to a marine lifestyle has been exceedingly rare. Major gene losses related to stomata, volatiles, defence and lignification are probably a consequence of the return to the sea rather than the cause of it. These new genomes will accelerate functional studies and solutions, as continuing losses of the 'savannahs of the sea' are of major concern in times of climate change and loss of biodiversity.
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Alismatales , Zosteraceae , Alismatales/genética , Zosteraceae/genética , EcosistemaRESUMEN
Most fresh bananas belong to the Cavendish and Gros Michel subgroups. Here, we report chromosome-scale genome assemblies of Cavendish (1.48 Gb) and Gros Michel (1.33 Gb), defining three subgenomes, Ban, Dh and Ze, with Musa acuminata ssp. banksii, malaccensis and zebrina as their major ancestral contributors, respectively. The insertion of repeat sequences in the Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 RGA2 (resistance gene analog 2) promoter was identified in most diploid and triploid bananas. We found that the receptor-like protein (RLP) locus, including Foc race 1-resistant genes, is absent in the Gros Michel Ze subgenome. We identified two NAP (NAC-like, activated by apetala3/pistillata) transcription factor homologs specifically and highly expressed in fruit that directly bind to the promoters of many fruit ripening genes and may be key regulators of fruit ripening. Our genome data should facilitate the breeding and super-domestication of bananas.
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Fusarium , Musa , Musa/genética , Fusarium/genética , Triploidía , Fitomejoramiento , Factores de Transcripción/genética , Enfermedades de las Plantas/genéticaRESUMEN
BACKGROUND: Sterile-fertile heteroblasty is a common phenomenon observed in ferns, where the leaf shape of a fern sporophyll, responsible for sporangium production, differs from that of a regular trophophyll. However, due to the large size and complexity of most fern genomes, the molecular mechanisms that regulate the formation of these functionally different heteroblasty have remained elusive. To shed light on these mechanisms, we generated a full-length transcriptome of Ceratopteris chingii with PacBio Iso-Seq from five tissue samples. By integrating Illumina-based sequencing short reads, we identified the genes exhibiting the most significant differential expression between sporophylls and trophophylls. RESULTS: The long reads were assembled, resulting in a total of 24,024 gene models. The differential expressed genes between heteroblasty primarily involved reproduction and cell wall composition, with a particular focus on expansin genes. Reconstructing the phylogeny of expansin genes across 19 plant species, ranging from green algae to seed plants, we identified four ortholog groups for expansins. The observed high expression of expansin genes in the young sporophylls of C. chingii emphasizes their role in the development of heteroblastic leaves. Through gene coexpression analysis, we identified highly divergent expressions of expansin genes both within and between species. CONCLUSIONS: The specific regulatory interactions and accompanying expression patterns of expansin genes are associated with variations in leaf shapes between sporophylls and trophophylls.
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Pared Celular , Fertilidad , Filogenia , Hojas de la Planta/genética , Reproducción , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
The importance of whole-genome duplication (WGD) for evolution is controversial. Whereas some view WGD mainly as detrimental and an evolutionary dead end, there is growing evidence that polyploidization can help overcome environmental change, stressful conditions, or periods of extinction. However, despite much research, the mechanistic underpinnings of why and how polyploids might be able to outcompete or outlive nonpolyploids at times of environmental upheaval remain elusive, especially for autopolyploids, in which heterosis effects are limited. On the longer term, WGD might increase both mutational and environmental robustness due to redundancy and increased genetic variation, but on the short-or even immediate-term, selective advantages of WGDs are harder to explain. Here, by duplicating artificially generated Gene Regulatory Networks (GRNs), we show that duplicated GRNs-and thus duplicated genomes-show higher signal output variation than nonduplicated GRNs. This increased variation leads to niche expansion and can provide polyploid populations with substantial advantages to survive environmental turmoil. In contrast, under stable environments, GRNs might be maladaptive to changes, a phenomenon that is exacerbated in duplicated GRNs. We believe that these results provide insights into how genome duplication and (auto)polyploidy might help organisms to adapt quickly to novel conditions and to survive ecological uproar or even cataclysmic events.
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Duplicación de Gen , Redes Reguladoras de Genes , Humanos , Genoma , Poliploidía , Evolución Molecular , Genoma de Planta/genéticaRESUMEN
MicroTom has a short growth cycle and high transformation efficiency, and is a prospective model plant for studying organ development, metabolism, and plant-microbe interactions. Here, with a newly assembled reference genome for this tomato cultivar and abundant RNA-seq data derived from tissues of different organs/developmental stages/treatments, we constructed multiple gene co-expression networks, which will provide valuable clues for the identification of important genes involved in diverse regulatory pathways during plant growth, e.g. arbuscular mycorrhizal symbiosis and fruit development. Additionally, non-coding RNAs, including miRNAs, lncRNAs, and circRNAs were also identified, together with their potential targets. Interacting networks between different types of non-coding RNAs (miRNA-lncRNA), and non-coding RNAs and genes (miRNA-mRNA and lncRNA-mRNA) were constructed as well. Our results and data will provide valuable information for the study of organ differentiation and development of this important fruit. Lastly, we established a database (http://eplant.njau.edu.cn/microTomBase/) with genomic and transcriptomic data, as well as details of gene co-expression and interacting networks on MicroTom, and this database should be of great value to those who want to adopt MicroTom as a model plant for research.
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Currents are unique drivers of oceanic phylogeography and thus determine the distribution of marine coastal species, along with past glaciations and sea-level changes. Here we reconstruct the worldwide colonization history of eelgrass (Zostera marina L.), the most widely distributed marine flowering plant or seagrass from its origin in the Northwest Pacific, based on nuclear and chloroplast genomes. We identified two divergent Pacific clades with evidence for admixture along the East Pacific coast. Two west-to-east (trans-Pacific) colonization events support the key role of the North Pacific Current. Time-calibrated nuclear and chloroplast phylogenies yielded concordant estimates of the arrival of Z. marina in the Atlantic through the Canadian Arctic, suggesting that eelgrass-based ecosystems, hotspots of biodiversity and carbon sequestration, have only been present there for ~243 ky (thousand years). Mediterranean populations were founded ~44 kya, while extant distributions along western and eastern Atlantic shores were founded at the end of the Last Glacial Maximum (~19 kya), with at least one major refuge being the North Carolina region. The recent colonization and five- to sevenfold lower genomic diversity of the Atlantic compared to the Pacific populations raises concern and opportunity about how Atlantic eelgrass might respond to rapidly warming coastal oceans.
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Ecosistema , Zosteraceae , Zosteraceae/genética , Canadá , Filogeografía , Océanos y MaresRESUMEN
Angiosperms have a complex history of whole-genome duplications (WGDs), with varying numbers and ages of WGD events across clades. These WGDs have greatly affected the composition of plant genomes due to the biased retention of genes belonging to certain functional categories following their duplication. In particular, regulatory genes and genes encoding proteins that act in multiprotein complexes have been retained in excess following WGD. Here, we inferred protein-protein interaction (PPI) networks and gene regulatory networks (GRNs) for seven well-characterized angiosperm species and explored the impact of both WGD and small-scale duplications (SSDs) in network topology by analyzing changes in frequency of network motifs. We found that PPI networks are enriched in WGD-derived genes associated with dosage-sensitive intricate systems, and strong selection pressures constrain the divergence of WGD-derived genes at the sequence and PPI levels. WGD-derived genes in network motifs are mostly associated with dosage-sensitive processes, such as regulation of transcription and cell cycle, translation, photosynthesis, and carbon metabolism, whereas SSD-derived genes in motifs are associated with response to biotic and abiotic stress. Recent polyploids have higher motif frequencies than ancient polyploids, whereas WGD-derived network motifs tend to be disrupted on the longer term. Our findings demonstrate that both WGD and SSD have contributed to the evolution of angiosperm GRNs, but in different ways, with WGD events likely having a more significant impact on the short-term evolution of polyploids.
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Redes Reguladoras de Genes , Magnoliopsida , Filogenia , Magnoliopsida/genética , Duplicación de Gen , Evolución Molecular , Genoma de Planta , PoliploidíaRESUMEN
When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes, and its only congeneric species, P. strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics, and the driving forces underneath the incipient speciation of the two Platycarya lineages.
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Carbonato de Calcio , Juglandaceae , Calcio , Especiación Genética , GenómicaRESUMEN
The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.