Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach.

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.

Hazard potential of Swiss Ixodes ricinus ticks: Virome composition and presence of selected bacterial and protozoan pathogens.

Ticks play an important role in transmitting many different emerging zoonotic pathogens that pose a significant threat to human and animal health. In Switzerland and abroad, the number of tick-borne diseases, in particular tick-borne encephalitis (TBE), has been increasing over the last few years. Thus, it remains essential to investigate the pathogen spectrum of ticks to rapidly detect emerging pathogens and initiate the necessary measures. To assess the risk of tick-borne diseases in different regions of Switzerland, we collected a total of 10'286 ticks from rural and urban areas in ten cantons in 2021 and 2022. Ticks were pooled according to species, developmental stage, gender, and collection site, and analyzed using next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). The metagenomic analysis revealed for the first time the presence of Alongshan virus (ALSV) in Swiss ticks. Interestingly, the pool-prevalence of ALSV was higher than that of tick-borne encephalitis virus (TBEV). Furthermore, several TBEV foci have been identified and pool prevalence of selected non-viral pathogens determined.

Resistance development in Escherichia coli to delafloxacin at pHs 6.0 and 7.3 compared to ciprofloxacin.

Understanding the resistance mechanisms of antibiotics in the micro-environment of the infection is important to assess their clinical applicability and potentially prevent resistance development. We compared the laboratory resistance evolution of Escherichia coli to delafloxacin (DLX) compared to ciprofloxacin (CIP), the co-resistance evolution, and underlying resistance mechanisms at different pHs. Three clones from each of the eight clinical E. coli isolates were subjected to subinhibitory concentrations of DLX or CIP in parallel at either pH 7.3 or 6.0. Minimum inhibitory concentrations (MICs) were regularly tested (at respective pHs), and the antibiotic concentration was adjusted accordingly. After 30 passages, MICs were determined in the presence of the efflux pump inhibitor phenylalanine-arginine-ß-naphthylamide. Whole genome sequencing of the parental isolates and their resistant derivatives (n = 54) was performed. Complementation assays were carried out for selected mutations. Quantitative PCR and efflux experiments were carried out for selected derivatives. For DLX-challenged strains, resistance to DLX evolved much slower in acidic than in neutral pH, whereas for CIP-challenged strains, the opposite was the case. Mutations in the quinolone resistance-determining region were mainly seen in CIP-challenged E. coli, whereas a multifactorial mechanism including mutations in efflux-related genes played a role in DLX resistance evolution (predominantly at pH 6.0). This work provides novel insights into the resistance mechanisms of E. coli to delafloxacin and highlights the importance of understanding micro-environmental conditions at the infection site that might affect the true clinical efficacy of antibiotics and challenges our current antibiotic susceptibility-testing paradigm.

Diagnostic performance of a new two-dimensional shear wave elastography expression using siemens ultrasound system combined with ACR TI-RADS for classification of benign and malignant thyroid nodules: A prospective multi-center study.

Objective: The present study aimed to evaluate the efficacy of a new two-dimensional shear wave elastography (2D-SWE) method using a Siemens ultrasound system and its combination with the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) for the differential diagnosis of benign and malignant thyroid nodules. Methods: Conventional ultrasound images and 2D-SWE (E-whole-mean and E-stiffest-mean) were prospectively analyzed in 593 thyroid nodules from 543 patients. Nodules were divided into diameter (D) ≤10 mm and D > 10 mm groups and graded using ACR TI-RADS. The receiver operating characteristic curve was plotted using pathological findings as the gold standard. Diagnostic performance was compared among 2D-SWE, ACR TI-RADS, and their combination. Results: The area under the curve (AUC) for E-whole-mean was higher than that for E-stiffest-mean (0.858 vs. 0.790, P < 0.001), which indicated that it was the better 2D-SWE parameter for differentiating malignant nodules from benign nodules with an optimal cut-off point of 11.36 kPa. In the all-sizes group, the AUC for E-whole-mean was higher than that for ACR TI-RADS (0.858 vs. 0.808, P < 0.001). The combination of E-whole-mean and ACR TI-RADS resulted in a higher AUC (0.929 vs. 0.858 vs. 0.808, P < 0.001), sensitivity (87.0% vs. 80.3% vs. 85.2%), specificity (85.1% vs. 74.0% vs. 73.6%), accuracy (86.3% vs. 78.1% vs. 81.1%), positive predictive value (91.5% vs. 85.1% vs. 85.6%), and negative predictive value (78.0% vs. 67.0% vs. 72.9%) compared to E-whole-mean or ACR TI-RADS alone. The AUC for the combination of 2D-SWE and ACR TI-RADS was superior to that for E-whole-mean or ACR TI-RADS alone in both D ≤ 10 mm and D > 10 mm groups (P < 0.001). Conclusion: As the better 2D-SWE parameter, E-whole-mean had a higher diagnostic power than ACR TI-RADS and enhanced the diagnostic performance of ACR TI-RADS when identifying benign and malignant thyroid nodules. The combination of E-whole-mean and ACR TI-RADS improved the diagnostic performance compared to using ACR TI-RADS alone, providing a new and reliable method for the clinical diagnosis of thyroid nodules.

Microbial dynamics in soils of the Damma glacier forefield show succession in the functional genetic potential.

Glacier retreat is a visible consequence of climate change worldwide. Although taxonomic change of the soil microbiomes in glacier forefields have been widely documented, how microbial genetic potential changes along succession is little known. Here, we used shotgun metagenomics to analyse whether the soil microbial genetic potential differed between four stages of soil development (SSD) sampled along three transects in the Damma glacier forefield (Switzerland). The SSDs were characterized by an increasing vegetation cover, from barren soil, to biological soil crust, to sparsely vegetated soil and finally to vegetated soil. Results suggested that SSD significantly influenced microbial genetic potential, with the lowest functional diversity surprisingly occurring in the vegetated soils. Overall, carbohydrate metabolism and secondary metabolite biosynthesis genes overrepresented in vegetated soils, which could be partly attributed to plant-soil feedbacks. For C degradation, glycoside hydrolase genes enriched in vegetated soils, while auxiliary activity and carbohydrate esterases genes overrepresented in barren soils, suggested high labile C degradation potential in vegetated, and high recalcitrant C degradation potential in barren soils. For N-cycling, organic N degradation and synthesis genes dominated along succession, and gene families involved in nitrification were overrepresented in barren soils. Our study provides new insights into how the microbial genetic potential changes during soil formation along the Damma glacier forefield.

Impact of ferroptosis on preeclampsia: A review.

Preeclampsia (PE) is usually associated with the accumulation of reactive oxygen species (ROS) resulting from heightened oxidative stress (OS). Ferroptosis is a unique type of lipid peroxidation-induced iron-dependent cell death distinct from traditional apoptosis, necroptosis, and pyroptosis and most likely contributes considerable to PE pathogenesis. At approximately 10-12 weeks of gestation, trophoblasts create an environment rich in oxygen and iron. In patients with PE, ferroptosis-related genes such as HIF1 and MAPK8 are downregulated, whereas PLIN2 is upregulated. Furthermore, miR-30b-5p overexpression inhibits solute carrier family 11 member 2, resulting in a decrease in glutathione levels and an increase in the labile iron pool. At the maternal-fetal interface, physiological hypoxia/reperfusion and excessive iron result in lipid peroxidation and ROS production. Owing to the high expression of Fpn and polyunsaturated fatty acid-containing phospholipid-related enzymes, including acyl-CoA synthetase long-chain family member 4, lysophosphatidylcholine acyl-transferase 3, and spermidine/spermine N1-acetyltransferase 1, trophoblasts become more susceptible to OS and ROS damage. In stage 1, the injured trophoblasts exhibit poor invasion and incomplete uterine spiral artery remodeling caused by ferroptosis, leading to placental ischemia and hypoxia. Subsequently, ferroptosis marked by OS occurs in stage 2, eventually causing PE. We aimed to explore the new therapeutic target of PE through OS in ferroptosis.

A High-Performance MoS2-Based Visible-Near-Infrared Photodetector from Gateless Photogating Effect Induced by Nickel Nanoparticles.

Recent advancements in two-dimensional materials have shown huge potential for optoelectronic applications. It is challenging to achieve highly effective and sensitive broadband photodetection based on MoS2 devices. Defect engineering, such as introducing vacancies, can narrow the bandgap and boost the separation of photogenerated carriers by defect states but leads to a slow response speed. Herein, we propose a nickel nanoparticle-induced gateless photogating effect with a unique energy band structure to enable the application of defect engineering and achieve high optoelectronic performance. The device based on Ni nanoparticle-decorated MoS2 with S vacancies exhibited high responsivities of 106.21 and 1.38 A W-1 and detectivities of 1.9 × 1012 and 8.9 × 109 Jones under 532 and 980 nm illumination (visible to near infrared), respectively, with highly accelerated response speed. This strategy provides new insight into optimizing defect engineering to design high-performance optoelectronic devices capable of broadband photodetection.

Distinct taxonomic and functional profiles of high Arctic and alpine permafrost-affected soil microbiomes.

BACKGROUND: Global warming is affecting all cold environments, including the European Alps and Arctic regions. Here, permafrost may be considered a unique ecosystem harboring a distinct microbiome. The frequent freeze-thaw cycles occurring in permafrost-affected soils, and mainly in the seasonally active top layers, modify microbial communities and consequently ecosystem processes. Although taxonomic responses of the microbiomes in permafrost-affected soils have been widely documented, studies about how the microbial genetic potential, especially pathways involved in C and N cycling, changes between active-layer soils and permafrost soils are rare. Here, we used shotgun metagenomics to analyze the microbial and functional diversity and the metabolic potential of permafrost-affected soil collected from an alpine site (Val Lavirun, Engadin area, Switzerland) and a High Arctic site (Station Nord, Villum Research Station, Greenland). The main goal was to discover the key genes abundant in the active-layer and permafrost soils, with the purpose to highlight the potential role of the functional genes found. RESULTS: We observed differences between the alpine and High Arctic sites in alpha- and beta-diversity, and in EggNOG, CAZy, and NCyc datasets. In the High Arctic site, the metagenome in permafrost soil had an overrepresentation (relative to that in active-layer soil) of genes involved in lipid transport by fatty acid desaturate and ABC transporters, i.e. genes that are useful in preventing microorganisms from freezing by increasing membrane fluidity, and genes involved in cell defense mechanisms. The majority of CAZy and NCyc genes were overrepresented in permafrost soils relative to active-layer soils in both localities, with genes involved in the degradation of carbon substrates and in the degradation of N compounds indicating high microbial activity in permafrost in response to climate warming. CONCLUSIONS: Our study on the functional characteristics of permafrost microbiomes underlines the remarkably high functional gene diversity of the High Arctic and temperate mountain permafrost, including a broad range of C- and N-cycling genes, and multiple survival and energetic metabolisms. Their metabolic versatility in using organic materials from ancient soils undergoing microbial degradation determine organic matter decomposition and greenhouse gas emissions upon permafrost thawing. Attention to their functional genes is therefore essential to predict potential soil-climate feedbacks to the future warmer climate.

Boron-Doping Induced Electron Delocalization in Fluorophosphate Cathode: Enhanced Na-Ion Diffusivity and Sodium-Ion Full Cell Performance.

Na3 V2 (PO4 )2 O2 F (NVPOF) is widely accepted as advanced cathode material for sodium-ion batteries with high application prospects ascribing to its considerable specific capacity and high working voltage. However, challenges in the full realization of its theoretical potential lie in the novel structural design to accelerate its Na+ diffusivity. Herein, considering the important role of polyanion groups in constituting Na+ diffusion tunnels, boron (B) is doped at the P-site to obtain Na3 V2 (P2- x Bx O8 )O2 F (NVP2- x Bx OF). As evidenced by density functional theory modeling, B-doping induces a dramatic decrease in the bandgap. Delocalization of electrons on the O anions in BO4 tetrahedra is observed in NVP2- x Bx OF, which dramatically lowers the electrostatic resistance experienced by Na+ . As a result, the Na+ diffusivity in the NVP2- x Bx OF cathode has accelerated up to 11 times higher, which secures a high rate property (67.2 mAh g-1 at 60 C) and long cycle stability (95.9% capacity retention at 108.6 mAh g-1 at 10 C after 1000 cycles). The assembled NVP1.90 B0.10 OF//Se-C full cell demonstrates exceptional power/energy density (213.3 W kg-1 @ 426.4 Wh kg-1 and 17970 W kg-1 @ 119.8 Wh kg-1 ) and outstanding capability to withstand long cycles (90.1% capacity retention after 1000 cycles at 105.3 mAh g-1 at 10 C).

Putative Signals of Generalist Plant Species Adaptation to Local Pollinator Communities and Abiotic Factors.

The reproductive success of flowering plants with generalized pollination systems is influenced by interactions with a diverse pollinator community and abiotic factors. However, knowledge about the adaptative potential of plants to complex ecological networks and the underlying genetic mechanisms is still limited. Based on a pool-sequencing approach of 21 natural populations of Brassica incana in Southern Italy, we combined a genome-environmental association analysis with a genome scan for signals of population genomic differentiation to discover genetic variants associated with the ecological variation. We identified genomic regions putatively involved in the adaptation of B. incana to the identity of local pollinator functional categories and pollinator community composition. Interestingly, we observed several shared candidate genes associated with long-tongue bees, soil texture, and temperature variation. We established a genomic map of potential generalist flowering plant local adaptation to complex biotic interactions, and the importance of considering multiple environmental factors to describe the adaptive landscape of plant populations.

The Pattern of RNA Editing Changes in Pleural Mesothelioma upon Epithelial-Mesenchymal Transition.

Pleural mesothelioma (PM) is a cancer where epithelioid, biphasic and sarcomatoid histotypes are observed. Sarcomatoid PM is characterized by mesenchymal features. Multi-omics have been used to characterize the epithelial-to-mesenchymal (EMT) phenotype at the molecular level. We contribute to this effort by including the analysis of RNA editing. We extracted samples with the highest vs. lowest Epithelial score from two PM cohorts and observed increased RNA editing in introns and decreased RNA editing in 3'UTR upon EMT. The same was observed in primary PM primary cultures stratified by transcriptomics analysis into two groups, one of them enriched with mesenchymal features. Our data demonstrate that, as has been observed in other cancer types, RNA editing associates to EMT phenotype in PM.

Viral Mimicry Response Is Associated With Clinical Outcome in Pleural Mesothelioma.

Introduction: The aim of this study was to investigate endogenous retrovirus (ERV) expression and type I interferon (IFN) activation in human pleural mesothelioma (PM) and their association with clinical outcome. Methods: The expression of ERV was determined from PM cohorts and mesothelial precursor RNA sequencing data. The expression of ERV was confirmed by quantitative polymerase chain reaction (qPCR). Methylation of genomic DNA was assessed by quantitative methylation-specific PCR. DNA demethylation was induced in cells by demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) treatment. To block type I IFN signaling, the cells were treated with ruxolitinib or MAVS silencing. The expression of IFN-stimulated genes (ISGs) was determined by qPCR and Western blot. Circulating ERVs were detected by qPCR. Results: Long terminal repeats (LTRs) represent the most abundant transposable elements up-regulated in PM. Within the LTR, ERVmap_1248 and LTR7Y, which are specifically enriched in PM, were further analyzed. The 5-Aza-CdR treatment increased the levels of ERVmap_1248 expression and induced ERVmap_1248 promoter demethylation in mesothelial cells. In addition, ERVmap_1248 promoter was more demethylated in the mesothelioma tissue compared with nontumor tissue. The 5-Aza-CdR treatment of the mesothelial cells also increased the levels of ISGs. Basal ISG expression was higher in the mesothelioma cells compared with the mesothelial cells, and it was significantly decreased by ruxolitinib treatment or MAVS silencing. Furthermore, ISG expression was higher in the tumor tissue with high expression levels of ERVmap_1248. High expression of ERVmap_1248 was associated with longer overall survival and BAP1 mutations. ERVmap_1248 and LTR7Y can be detected in the PM plasma. Conclusions: We provide clues for patient stratification especially for immunotherapy where best clinical responses are associated with an activated basal immune response.

Long-term mercury contamination does not affect the microbial gene potential for C and N cycling in soils but enhances detoxification gene abundance.

Soil microorganisms are key transformers of mercury (Hg), a toxic and widespread pollutant. It remains uncertain, however, how long-term exposure to Hg affects crucial microbial functions, such as litter decomposition and nitrogen cycling. Here, we used a metagenomic approach to investigate the state of soil functions in an agricultural floodplain contaminated with Hg for more than 80 years. We sampled soils along a gradient of Hg contamination (high, moderate, low). Hg concentrations at the highly contaminated site (36 mg kg-1 dry soil on average) were approximately 10 times higher than at the moderately contaminated site (3 mg kg-1 dry soil) and more than 100 times higher than at the site with low contamination (0.25 mg kg-1 dry soil; corresponding to the natural background concentration in Switzerland). The analysis of the CAZy and NCyc databases showed that carbon and nitrogen cycling was not strongly affected with high Hg concentrations, although a significant change in the beta-diversity of the predicted genes was observed. The only functional classes from the CAZy database that were significantly positively overrepresented under higher Hg concentrations were genes involved in pectin degradation, and from the NCyc database dissimilatory nitrate reduction and N-fixation. When comparing between low and high Hg concentrations the genes of the EggNOG functional category of inorganic ion transport and metabolism, two genes encoding Hg transport proteins and one gene involved in heavy metal transport detoxification were among those that were highly significantly overrepresented. A look at genes specifically involved in detoxification of Hg species, such as the mer and hgc genes, showed a significant overrepresentation when Hg contamination was increased. Normalized counts of these genes revealed a dominant role for the phylum Proteobacteria. In particular, most counts for almost all mer genes were found in Betaproteobacteria. In contrast, hgc genes were most abundant in Desulfuromonadales. Overall, we conclude from this metagenomic analysis that long-term exposure to high Hg triggers shifts in the functional beta-diversity of the predicted microbial genes, but we do not see a dramatic change or breakdown in functional capabilities, but rather functional redundancy.

Heterogeneous RNA editing and influence of ADAR2 on mesothelioma chemoresistance and the tumor microenvironment.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. We found that tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3'UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment, and type-1 interferon signaling upregulation, leading to changes in the microenvironment in vivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also in chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

Virus Diversity, Abundance, and Evolution in Three Different Bat Colonies in Switzerland.

Bats are increasingly recognized as reservoirs for many different viruses that threaten public health, such as Hendravirus, Ebolavirus, Nipahvirus, and SARS- and MERS-coronavirus. To assess spillover risk, viromes of bats from different parts of the world have been investigated in the past. As opposed to most of these prior studies, which determined the bat virome at a single time point, the current work was performed to monitor changes over time. Specifically, fecal samples of three endemic Swiss bat colonies consisting of three different bat species were collected over three years and analyzed using next-generation sequencing. Furthermore, single nucleotide variants of selected DNA and RNA viruses were analyzed to investigate virus genome evolution. In total, sequences of 22 different virus families were found, of which 13 are known to infect vertebrates. Most interestingly, in a Vespertilio murinus colony, sequences from a MERS-related beta-coronavirus were consistently detected over three consecutive years, which allowed us to investigate viral genome evolution in a natural reservoir host.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Differential Viral Genome Diversity of Healthy and RSS-Affected Broiler Flocks.

The intestinal virus community contributes to health and disease. Runting and stunting syndrome (RSS) is associated with enteric viruses and leads to economic losses in the poultry industry. However, many viruses that potentially cause this syndrome have also been identified in healthy animals. To determine the difference in the virome of healthy and diseased broilers, samples from 11 healthy and 17 affected broiler flocks were collected at two time points and analyzed by Next-Generation Sequencing. Virus genomes of Parvoviridae, Astroviridae, Picornaviridae, Caliciviridae, Reoviridae, Adenoviridae, Coronaviridae, and Smacoviridae were identified at various days of poultry production. De novo sequence analysis revealed 288 full or partial avian virus genomes, of which 97 belonged to the novel genus Chaphamaparvovirus. This study expands the knowledge of the diversity of enteric viruses in healthy and RSS-affected broiler flocks and questions the association of some viruses with the diseases.

High-Throughput Calculation of Interlayer van der Waals Forces Validated with Experimental Measurements.

Interlayer van der Waals interactions play an important role in two-dimensional (2D) materials on various occasions. The interlayer binding force is often directly measured and is considered more closely related to the exfoliation condition. However, a binding force database from accurate theoretical calculations does not yet exist. In this work, the critical interlayer binding force and energy are directly calculated for 230 2D materials, which exhibit divergent trends. A linear relationship that links the two quantities with the equilibrium interlayer distance is found and checked. Experiments are carried out for three different materials using atomic force microscopy. The measured forces show a consistent trend with the calculated results, and the estimated binding strengths are of the same order of magnitude as the predicted values. Our work can provide a reliable reference for interlayer adhesion studies and help establish accurate models of exfoliation processes.

The haplotype-resolved chromosome pairs of a heterozygous diploid African cassava cultivar reveal novel pan-genome and allele-specific transcriptome features.

BACKGROUND: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome. FINDINGS: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. CONCLUSIONS: The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.