Search

Background: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. Results: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. Conclusions: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants. [ABSTRACT FROM AUTHOR]

DNA methylation is a highly conserved epigenetic modification involved in many biological processes, including growth and development, stress response, and secondary metabolism. DNA demethylase (DNA-deMTase) genes have been identified in some plant species; however, there are no reports on the identification and analysis of DNA-deMTase genes in Foxtail millet (Setaria italica L.). In this study, seven DNA-deMTases were identified in S. italica. These DNA-deMTase genes were divided into four subfamilies (DML5, DML4, DML3, and ROS1) by phylogenetic and gene structure analysis. Further analysis shows that the physical and chemical properties of these DNA-deMTases proteins are similar, contain the typical conserved domains of ENCO3c and are located in the nucleus. Furthermore, multiple cis-acting elements were observed in DNA-deMTases, including light responsiveness, phytohormone responsiveness, stress responsiveness, and elements related to plant growth and development. The DNA-deMTase genes are expressed in all tissues detected with certain tissue specificity. Then, we investigated the abundance of DNA-deMTase transcripts under abiotic stresses (cold, drought, salt, ABA, and MeJA). The results showed that different genes of DNA-deMTases were involved in the regulation of different abiotic stresses. In total, our findings will provide a basis for the roles of DNA-deMTase in response to abiotic stress. [ABSTRACT FROM AUTHOR]

Dietary analysis in wildlife is fundamental for understanding their flexible response to seasonal changes and developing effective conservation management measures. Taihangshan macaque (Macaca mulatta tcheliensis) is the northernmost population of rhesus macaque, currently only distributed in the southern Mt. Taihangshan area. This area belongs to a semi‐arid region resulting in limited plant food availability for Taihangshan macaques, with seasonal variation. Herein, we used a chloroplast trnL DNA metabarcoding approach to identify the plant diet diversity and composition from 100 fecal samples of Taihangshan macaque in four seasons (spring, summer, autumn, and winter) from 2020 to 2021. The results revealed that (1) a total of 48 distinct families, 88 genera, and 52 species within the 105 food items that were consumed by Taihangshan macaques throughout the year; (2) the diversity of food items exhibited significant differences across the four seasons; (3) Rosaceae, Rhamnaceae, Fagaceae, and Poaceae are the preferential food items for Taihangshan macaques and have different relative abundances, fluctuating with seasonal variation. DNA metabarcoding can expand our understanding of the repertoire of food items consumed by Taihangshan macaques by detecting some consumed food items in this population that were not yet discovered using traditional methods. Therefore, the integrative results from traditional methods and DNA metabarcoding can provide a fundamental understanding of dietary composition to guide the conservation management of Taihangshan macaques. [ABSTRACT FROM AUTHOR]

Decades of empirical research have revealed how the geological history of our planet shaped plant evolution by establishing well‐known patterns (e.g., how mountain uplift resulted in high rates of diversification and replicate radiations in montane plant taxa). This follows a traditional approach where botanical data are interpreted in light of geological events. In this synthesis, I instead describe how by integrating natural history, phylogenetics, and population genetics, botanical research can be applied alongside geology and paleontology to inform our understanding of past geological and climatic processes. This conceptual shift aligns with the goals of the emerging field of geogenomics. In the neotropics, plant geogenomics is a powerful tool for the reciprocal exploration of two long standing questions in biology and geology: how the dynamic landscape of the region came to be and how it shaped the evolution of the richest flora. Current challenges that are specific to analytical approaches for plant geogenomics are discussed. I describe the scale at which various geological questions can be addressed from biological data and what makes some groups of plants excellent model systems for geogenomics research. Although plant geogenomics is discussed with reference to the neotropics, the recommendations given here for approaches to plant geogenomics can and should be expanded to exploring long‐standing questions on how the earth evolved with the use of plant DNA. [ABSTRACT FROM AUTHOR]

Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages in some applications. We identified 54,837 CRISPR‐Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single‐ and mixed‐species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed‐species experiments, single‐species experiments yielded more on‐target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed‐species experiments yielded sufficient data to provide ≥48‐fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL‐P6 marker. Prior work developed CRISPR‐based enrichment protocols for long‐read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short‐read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR‐based analyses of mixed‐species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori. [ABSTRACT FROM AUTHOR]

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation pathway that relies on the atypical RNA polymerases Pol IV-RDR2 complex and Pol V to produce the siRNA precursor and lncRNA, respectively. Both Pol IV and Pol V have evolved from Pol II, but adopt unique biochemical features relevant for their functions in siRNA precursor (Pol IV) and scaffold lncRNA (Pol V) production. Recent structural studies suggest that the Pol IV and Pol V NRPD(E)2 subunit-induced enhanced backtracking plays an important role in their ncRNA production function. A dual measurement mechanism was proposed for the siRNA precursor production by Pol IV-RDR2 holoenzyme in RdDM. In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM. [ABSTRACT FROM AUTHOR]

Microbes can overcome the ability of plant resistance genes to confer protection against disease by mutating their corresponding avirulence genes. The fungus Leptosphaeria maculans causes blackleg disease on canola crops and subverts Brassica napus resistance genes through several DNA mutation mechanisms. One of these is repeat‐induced point (RIP) mutation, which can 'leak' into the avirulence genes from the adjacent repetitive sequences that the mutation process is targeting. Here, we identified populations of L. maculans in Australia that have extensive RIP mutations in the avirulence gene AvrLm2 and show that this has been triggered by a duplication of the gene and surrounding DNA that includes the distant (>55 kb in total) AvrLm6 gene. This finding provides another mechanism of mutation by which pathogens can overcome host resistance, and more broadly contributes to understanding the complex balance between gene duplication and genome defence. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is a xylem‐invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1‐Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild‐type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP‐overexpressing transformant was 22 times higher than that in plants infected with the wild‐type isolate, further supporting the critical role of VdNEP in infection. A VdNEP‐EGFP fusion was constructed to follow its localization in fungal cells and during infection. [ABSTRACT FROM AUTHOR]

The increasing number of plant mitochondrial DNA genomes (mtDNA) sequenced reveals the extent of transfer from both chloroplast DNA genomes (cpDNA) and nuclear DNA genomes (nDNA). This study created a library and assembled the chloroplast and mitochondrial genomes of the leafy sweet potato better to understand the extent of mitochondrial and chloroplast gene transfer. The full-length chloroplast genome of the leafy sweet potato (OM808940) is 161,387 bp, with 132 genes annotated, including 87 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. The mitochondrial genome (OM808941) was 269,578 bp in length and contained 69 functional genes, including 39 protein-coding genes, 6 rRNA genes, and 24 tRNA genes. 68 SSR loci were found in the leafy sweet potato organelle genome, including 54 in the chloroplast genome and 14 in the mitochondria genome. In the sweet potato mitochondrial genome, most genes have RNA editing sites, and the conversion ratio from hydrophilic amino acids to hydrophobic amino acids is the highest, reaching 47.12%. Horizontal transfer occurs in the sweet potato organelle genome and nuclear genome. 40 mitochondrial genome segments share high homology with 14 chloroplast genome segments, 33 of which may be derived from chloroplast genome horizontal transfer. 171 mitochondrial genome sequences come from the horizontal transfer of nuclear genome. The phylogenetic analysis of organelle genes revealed that the leafy sweet potato was closely related to the tetraploid wild species Ipomoea tabascana and the wild diploid species Ipomoea trifida. [ABSTRACT FROM AUTHOR]

As the social organism, Polistes wasps build a communal nest using woody fibers with saliva for sustaining brood and adult population throughout the season. Limited information exists regarding the identification specific plant materials employed in wasp nest building. Thus, we firstly tested if the DNA metabarcoding approach utilizing rbcL and trnL molecular markers could identify the plant species quantitatively and qualitatively inform the mixed-origin woody samples. A threshold of 0.01 proportion of reads was applied for rbcL and trnL molecular markers, while this threshold for median proportion was 0.0025. In assessing taxa richness, the median proportion demonstrated superior performance, exhibiting higher taxa detection power, however, rbcL marker outperformed in quantitative analysis. Subsequently, we applied DNA metabarcoding to identify the plant materials from the nests of two Polistes species, P. mandarinus and P. rothneyi. The results showed that higher preference of Quercus and Robinia as the major nest building materials regardless of the surrounding plant communities, by two wasp species. Material diversity was higher for P. rothneyi than P. mandarinus, which may explain the abundance of this species possibly with heightened adaptive capacities in their nesting behavior. This study demonstrated that DNA metabarcoding could identify the complex nest-building plant materials of paper wasps and provide insights into their ecological interactions in the natural ecosystem. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the utilization of a buffer solution, containing Tris, HCl, EDTA, NaCl, and glycerol (THENG), in combination with a thermal shock process, as a novel DNA extraction method (THENG-tsp) for endophytic fungi. Three mg of fungal mycelium was added to 100 μL of THENG buffer and incubated in a water-bath (83 ± 2 °C) for 35 min. The vials were subsequently frozen at -20 °C for 15 min and then subjected to centrifugation (11,200 m/s2; 6 min). The resulting supernatant was used as a pure DNA source. In addition, the DNA of endophytic fungi was extracted using the cetyl trimethylammonium bromide (CTAB) method. DNA quality was evaluated based on light intensity and distance (in pixels) of bands observed on a 0.8% agarose gel after electrophoresis. The results indicate that the application of the THENG-tsp method improved the average band quality of all endophytic fungi by 7.86%. More precisely, bands corresponding to endophytic fungi belonging to the Basidiomycota and Ascomycota phyla exhibited an average increase in band quality of 13.50% and 8.57%, respectively, compared to the CTAB method. The application of the THENG-tsp method also resulted in improved band quality for Alternaria spp. and Fusarium spp., with average increases of 8.47% and 28.40%, respectively, compared to the CTAB method. Hence, the THENG-tsp method is considered appropriate for DNA extraction from endophytic fungi in laboratory settings due to its minimal requirement of initial fungal mycelium, time efficiency, and the absence of the use of potentially harmful chemical compounds. [ABSTRACT FROM AUTHOR]

Thirty years ago, DNA sequences were obtained from an extinct Aotearoa New Zealand animal for the first time. Since then, ancient DNA research has provided many – often unexpected – insights into the origins of New Zealand's terrestrial and marine vertebrate fauna. Because recent human activities in New Zealand have caused the decline or extinction of many endemic plant, bird, reptile, and marine mammal species, ancient DNA has been instrumental in reconstructing their identities and origins. However, most ancient DNA studies focusing on New Zealand species have been restricted to vertebrates, with small sample sizes, and/or relatively few genetic markers. This has limited their power to infer fine-scale biogeographic patterns, including (pre)historic distributions and range-shifts driven by past climate and environmental change. Recently, 'next-generation' methodological and technological advances have broadened the range of hypotheses that can feasibly be tested with ancient DNA. These advances represent an exciting opportunity for further exploring New Zealand biogeography using ancient DNA, but their promise has not yet been fully realised. In this review, we summarise the last 30 years of ancient DNA research into New Zealand faunal biogeography and highlight key objectives, challenges, and possibilities for the next 30 years and beyond. [ABSTRACT FROM AUTHOR]

SUMMARY: Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium‐mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant‐microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long‐read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium‐mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large‐scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants. Significance Statement: Agrobacterium‐mediated transformation (AMT) of plants is used extensively as a tool to create new genetic variants for research purposes or agricultural needs. In this work, we summarize the molecular mechanisms involved in AMT and the effects on plant genomes resulting from the integration of exogenous DNA through this method. [ABSTRACT FROM AUTHOR]

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18–25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment. [ABSTRACT FROM AUTHOR]

Background: Beyond the massive amounts of DNA and genes transferred from the protoorganelle genome to the nucleus during the endosymbiotic event that gave rise to the plastids, stretches of plastid DNA of varying size are still being copied and relocated to the nuclear genome in a process that is ongoing and does not result in the concomitant shrinking of the plastid genome. As a result, plant nuclear genomes feature small, but variable, fraction of their genomes of plastid origin, the so-called nuclear plastid DNA sequences (NUPTs). However, the mechanisms underlying the origin and fixation of NUPTs are not yet fully elucidated and research on the topic has been mostly focused on a limited number of species and of plastid DNA. Results: Here, we leveraged a chromosome-scale version of the genome of the orphan crop Moringa oleifera, which features the largest fraction of plastid DNA in any plant nuclear genome known so far, to gain insights into the mechanisms of origin of NUPTs. For this purpose, we examined the chromosomal distribution and arrangement of NUPTs, we explicitly modeled and tested the correlation between their age and size distribution, we characterized their sites of origin at the chloroplast genome and their sites of insertion at the nuclear one, as well as we investigated their arrangement in clusters. We found a bimodal distribution of NUPT relative ages, which implies NUPTs in moringa were formed through two separate events. Furthermore, NUPTs from every event showed markedly distinctive features, suggesting they originated through distinct mechanisms. Conclusions: Our results reveal an unanticipated complexity of the mechanisms at the origin of NUPTs and of the evolutionary forces behind their fixation and highlight moringa species as an exceptional model to assess the impact of plastid DNA in the evolution of the architecture and function of plant nuclear genomes. [ABSTRACT FROM AUTHOR]

Molecular breeding methods, such as marker-assisted selection and genomic selection, require high-throughput and cost-effective methods for isolating genomic DNA from plants, specifically from crop tissue or seed with high polysaccharides, lipids, and proteins. A quick and inexpensive high-throughput method for isolating genomic DNA from seed and leaf tissue from multiple crops was tested with a DNA isolation method that combines CTAB extraction buffer and lab-made SA-coated magnetic nanoparticles. This method is capable of isolating quality genomic DNA from leaf tissue and seeds in less than 2 hours with fewer steps than a standard CTAB extraction method. The yield of the genomic DNA was 582–729 ng per 5 leaf discs or 216–1869 ng per seed in soybean, 2.92–62.6 ng per 5 leaf discs or 78.9–219 ng per seed in wheat, and 30.9–35.4 ng per 5 leaf discs in maize. The isolated DNA was tested with multiple molecular breeding methods and was found to be of sufficient quality and quantity for PCR and targeted genotyping by sequencing methods such as molecular inversion probes (MIPs). The combination of SA-coated magnetic nanoparticles and CTAB extraction buffer is a fast, simple, and environmentally friendly, high-throughput method for both leaf tissues and seed(s) DNA preparation at low cost per sample. The DNA obtained from this method can be deployed in applied breeding programs for marker-assisted selection or genomic selection. [ABSTRACT FROM AUTHOR]

Background: Viral diseases continue to pose a major threat to the world's commercial crops. The in-depth exploration and efficient utilization of resistance proteins have become crucial strategies for their control. However, current delivery methods for introducing foreign DNA suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. The nanocarriers provides a convenient channel for the DNA delivery and functional utilization of disease-resistant proteins. Results: In this research, we identified a cysteine-rich venom protein (NbCRVP) in Nicotiana benthamiana for the first time. Virus-induced gene silencing and transient overexpression clarified that NbCRVP could inhibit the infection of tobacco mosaic virus, potato virus Y, and cucumber mosaic virus, making it a broad-spectrum antiviral protein. Yeast two-hybrid assay, co-immunoprecipitation, and bimolecular fluorescence complementation revealed that calcium-dependent lipid-binding (CaLB domain) family protein (NbCalB) interacted with NbCRVP to assist NbCRVP playing a stronger antiviral effect. Here, we demonstrated for the first time the efficient co-delivery of DNA expressing NbCRVP and NbCalB into plants using poly(amidoamine) (PAMAM) nanocarriers, achieving stronger broad-spectrum antiviral effects. Conclusions: Our work presents a tool for species-independent transfer of two interacting protein DNA into plant cells in a specific ratio for enhanced antiviral effect without transgenic integration, which further demonstrated new strategies for nanocarrier-mediated DNA delivery of disease-resistant proteins. [ABSTRACT FROM AUTHOR]

Understanding trophic interactions in agroecosystems is crucial for harnessing ecosystem services such as pest control, thus enabling a reduction in pesticide use. Carabid beetles (Coleoptera: Carabidae) have the potential to regulate not only insect pests but also weed seeds and slugs. The aim of this study was to investigate the food choice of different carabid species in the experimental setting of a cereal field with varying seed and slug prey availability during the season. In addition to varying food availability, the effects of species identity and season on carabid food choice should also be closely examined. Therefore, the gut contents of 1,120 beetles of eight carabid species were screened for the DNA of plants, aphids, springtails, earthworms and slugs via diagnostic multiplex PCR and a nested metabarcoding approach for plant species identification. Plant DNA was detected far more often (72%) than the various animal prey types (less than 12.5% each). Within the plant detections, 80 weed species were identified in the metabarcoding, with Galinsoga parviflora/quadriradiata (Galinsoga spp.—quickweeds) as the most frequently detected species. Carabid food choice was driven by their species identity and seasonality, while no effect of increased availability of seeds and slugs on their food choice was detected. While weed seeds seem to be an important food source for carabids, their availability does not directly affect the carabid diet. The importance of consumer identity and seasonality highlight the need for a diverse carabid species community for resilient pest control services. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms, such as DNA methylation, can influence gene regulation and affect phenotypic variation, raising the possibility that they contribute to ecological adaptation. Beginning to address this issue requires high‐resolution sequencing studies of natural populations to pinpoint epigenetic regions of potential ecological and evolutionary significance. However, such studies are still relatively uncommon, especially in insects, and are mainly restricted to a few model organisms. Here, we characterize patterns of DNA methylation for natural populations of Timema cristinae adapted to two host plant species (i.e. ecotypes). By integrating results from sequencing of whole transcriptomes, genomes and methylomes, we investigate whether environmental, host and genetic differences of these stick insects are associated with methylation levels of cytosine nucleotides in the CpG context. We report an overall genome‐wide methylation level for T. cristinae of ~14%, with methylation being enriched in gene bodies and impoverished in repetitive elements. Genome‐wide DNA methylation variation was strongly positively correlated with genetic distance (relatedness), but also exhibited significant host‐plant effects. Using methylome‐environment association analysis, we pinpointed specific genomic regions that are differentially methylated between ecotypes, with these regions being enriched for genes with functions in membrane processes. The observed association between methylation variation and genetic relatedness, and with the ecologically important variable of host plant, suggests a potential role for epigenetic modification in T. cristinae adaptation. To substantiate such adaptive significance, future studies could test whether methylation can be transmitted across generations and the extent to which it responds to experimental manipulation in field and laboratory studies. [ABSTRACT FROM AUTHOR]

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15–100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis. [ABSTRACT FROM AUTHOR]

Feeding on plant-derived sugars is an essential component of mosquito biology, affecting key aspects of their lives such as survival, metabolism, and reproduction. Among mosquitoes, Aedes aegypti and Aedes albopictus are two invasive mosquito species in the US, and are vectors of diseases such as dengue fever, chikungunya, and Zika. These species live in heavily populated, urban areas, where they have high accessibility to human hosts as well as to plants in backyards and public landscapes. However, the range of plants that are suitable sugar hosts for these species remains to be described, despite the importance of understanding what plants may attract or repel mosquitoes to inform citizens and municipal authorities accordingly. Here, we tested whether Ae. aegypti and Ae. albopictus would sugar-feed on eleven commonly planted ornamental plant species. We confirmed feeding activity using the anthrone method and identified the volatile composition of plant headspace using gas-chromatography mass-spectroscopy. These chemical analyses revealed that a broad range of olfactory cues are associated with plants that mosquitoes feed on. This prompted us to use plant DNA barcoding to identify plants that field-caught mosquitoes feed on. Altogether, results show that native and invasive mosquito species can exploit a broader range of plants than originally suspected, including wild and ornamental plants from different phyla throughout the Spring, Summer and Fall seasons. [ABSTRACT FROM AUTHOR]

Sample preservation often impedes efforts to generate high-quality reference genomes or pangenomes for Earth's more than 2 million plant and animal species due to nucleotide degradation. Here we compare the impacts of storage methods including solution type, temperature, and time on DNA quality and Oxford Nanopore long-read sequencing quality in 9 fish and 4 plant species. We show 95% ethanol largely protects against degradation for fish blood (22 °C, ≤6 weeks) and plant tissue (4 °C, ≤3 weeks). From this furthest storage timepoint, we assemble high-quality reference genomes of 3 fish and 2 plant species with contiguity (contig N50) and completeness (BUSCO) that achieve the Vertebrate Genome Project benchmarking standards. For epigenetic applications, we also report methylation frequency compared to liquid nitrogen control. The results presented here remove the necessity for cryogenic storage in many long read applications and provide a framework for future studies focused on sampling in remote locations, which may represent a large portion of the future sequencing of novel organisms. Long-read sequencing of fish and plant samples demonstrate that ethanol suitably preserves field-collected specimens for genome assembly of organisms, reducing the necessity for cryogenic storage. [ABSTRACT FROM AUTHOR]

Background: Plant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant. Results: We present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode. Conclusion: We were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes. [ABSTRACT FROM AUTHOR]

Zingiber loerzingii Valeton is one of the species in the Zingiberaceae family found throughout Aceh and North Sumatra, Indonesia, with slimy flowers, yellowish white color, and dark orange stamens. Z. loerzingii is endemic in North Sumatra with a very limited distribution. The International Union for Conservation of Nature and Natural Resources classifies this plant into the vulnerable ones category. This study aims to examine the potential of DNA barcoding from nuclear DNA (ITS2) and DNA chloroplasts (rbcL and trnL-F) to identify Z. loerzingii plants. The research sample was obtained from two main distribution areas of Z. loerzingii in North Sumatra, Indonesia, namely Sibolangit Nature Reserve and Tangkahan Conservation Forest. The results showed that all the DNA barcode markers used were able to classify Z. loerzingii into the same group in the phylogenetic analysis. ITS marker is the most effective marker for classifying Zingiberaceae species compared to rbcL and trnL-F markers. The ITS2 marker has the lowest level of intraspecific and intraspecific genetic distance overlap compared to the rbcL and trnL-F markers. This research is expected to provide information related to the DNA barcode of Z. loerzingii in an effort to conserve this rare plant. [ABSTRACT FROM AUTHOR]

This study uses morphological characteristics and RAPD markers to evaluate the polyploidization of synthetic porang. Seeds of triploid porang (2n=2x=26) were soaked in the different colchicine concentrations for 24 hours. After colchicine treatment, the porang seeds were planted to an MS medium that contained 2.2 µM of 6-benzylaminopurine (BAP), then, 40 days after planting in the MS media, the morphology and molecular of synthetic polyploid porang were characterized. For DNA extraction, a total of 100 mg of young leaves of porang plantlet was collected. One way Anova followed by the Duncan test (95%) was performed for phenotypic characterization. The number of different alleles, number of effective alleles, Shannon's information index, diversity, and unbiased diversity were assessed for genetic diversity. Synthetic polyploid porang has a higher total shoot, root, and wider leaves than normal porang. Polyploidy induction also successfully increased the genetic diversity of porang, and the genetic diversity will increase porang adaptability and sustainability of porang cultivation. [ABSTRACT FROM AUTHOR]

This study assesses the mechanism of action of plant-based silver nanoparticles (AgNPs) against antibiotic-resistant bacteria. We compared AgNPs synthesized through Salvia moorcroftiana and Origanum vulgare extracts and their conjugates with the antibiotic Ceftriaxone for their capacity to cause oxidative damage through reactive oxygen species (ROS). We quantified ROS in the cells of two bacterial strains after treating them with all AgNP types and observed that AgNPs were most effective in K. pneumoniae as they resulted in the highest ChS1 count (44,675), while in P. aeruginosa, Cfx-AgNPs induced the highest levels of ROS with ChS1 count of 56,865. DNA analysis showed that both plant-based AgNPs (O-AgNPs = 0.192 and S-AgNPs = 0.152) were most effective in K. pneumoniae and S-AgNPs (abs = 0.174) and O-Cfx-AgNPs (abs = 0.261) in P. aeruginosa. We observed a significant increase in the levels of conjugated dienes (86.4 μM) and malondialdehyde (172.25 nM) in the bacterial strains after treatment with AgNPs, compared to the control (71.65 μM and 18.064 nM, respectively, in K. pneumoniae and P. aeruginosa). These results indicate lipid peroxidation. AgNPs also increased the levels of protein thiols (0.672 nM) compared to the control (0.441 nM) in K. pneumoniae, except for Chem-AgNPs (0.21 nM). These results suggest that plant-based AgNPs are more effective in oxidizing bacterial DNA, protein, and lipids than Chem-AgNPs. Furthermore, protein oxidation varied between AgNPs alone and AgNPs-antibiotic conjugates. The highest levels of protein thiols were found in the samples treated with O-Cfx-AgNPs (0.672 nM and 0.525 nM in K. pneumoniae and P. aeruginosa, respectively). The results demonstrated that AgNPs kill bacteria by altering bacterial macromolecules such as DNA, lipids, and proteins. [ABSTRACT FROM AUTHOR]

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is driven by small RNAs (sRNAs) complementary to the nascent transcript of RNA polymerase V (Pol V). sRNAs associated with ARGONAUTE (AGO) proteins are tethered to Pol V mainly by the AGO-hook domain of its subunit NRPE1. We found, by in silico analyses, that Pol V strongly colocalizes on chromatin with another AGO-hook protein, SPT6-like (SPT6L), which is a known essential transcription elongation factor of Pol II. Our phylogenetic analysis revealed that SPT6L acquired its AGO-binding capacity already in the most basal streptophyte algae, even before the emergence of Pol V, suggesting that SPT6L might be a driving force behind the RdDM evolution. Since its emergence, SPT6L with the AGO-hook represents the only conserved SPT6 homolog in Viridiplantae, implying that the same protein is involved in both Pol II and Pol V complexes. To better understand the role of SPT6L in the Pol V complex, we characterized genomic loci where these two colocalize and uncovered that DNA methylation there is more dynamic, driven by higher levels of sRNAs often from non-canonical RdDM pathways and more dependent on chromatin modifying and remodeling proteins like MORC. Pol V loci with SPT6L are highly depleted in helitrons but enriched in gene promoters for which locally and temporally precise methylation is necessary. In view of these results, we discuss potential roles of multiple AGO-hook domains present in the Pol V complex and speculate that SPT6L mediates de novo methylation of naïve loci by interconnecting Pol II and Pol V activities. [ABSTRACT FROM AUTHOR]

Honey is well-known as a food product that is rich in active ingredients and is very popular among consumers. Free amino acids (FAAs) are one of the important nutritional components of honey, which can be used not only as a nutritional indicator of honey but also as an indicator of plant source identification. In this study, the contents of 20 FAAs in seven types of honey from 11 provinces in China were examined for the first time. The 20 FAAs were analyzed by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS). By analyzing 93 honey samples from seven types of honey, the FAAs were found to range from 394.4 mg/kg (linden honey) to 1771.7 mg/kg (chaste honey). Proline ranged from 274.55 to 572.48 mg/kg, and methionine was only present in some of the linden honey, chaste honey, acacia honey, and rape honey. Evaluated by amino acid principal component analysis, multifloral grassland honey had the highest overall evaluation score, acacia and jujube honey were the most similar, while chaste honey was the least similar to the other types of honey. In addition, DNA was extracted from 174 Xinjiang grassland honey samples and different plant leaves for PCR and sequencing to identify the species of nectar plants. As a result, 12 families and 25 species of honey plants were identified. The results confirmed the diversity of FAAs in dissimilar types and sources of honey. This study provides a reference for expanding honey quality standards and verifying the authenticity of honey. [ABSTRACT FROM AUTHOR]

DNA methylation is a kind of epigenetic modification regulated by DNA methyltransferases and DNA demethylases. Here, the influences of DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) combined with plant hormones [abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA3)] and small gaseous signal molecules [(nitric oxide (NO), hydrogen sulfide (H2S), and hydrogen gas (H2)] on the tomato (Solanum lycopersicum) seedlings growth were explored. Moreover, the expression levels of DNA methyltransferases and demethylases under plant hormones and small gaseous molecules were detected. We found that ABA and MeJA restrained tomato seedling growth, and GA3, NO, H2S, and H2 promoted seedling growth. Nevertheless, 5-azadC treatment retarded the effects of these plant growth regulators mentioned above. Moreover, ABA boosted SlDRM5 expression, whereas GA3 and MeJA treatments inhibited the expression. Sodium hydrosulfide and hydrogen-rich water treatments affected SlDRM5 expression. Moreover, MeJA increased the quantity of SlDML1 transcripts from 3 to 24 h, while other therapies decreased the quantity. It is interesting to note that GA3 expressively induced SlMET1 and SlDML2 expression. Hence, DNA methylation may be essential for plant growth and is controlled by phytohormones and gaseous signal channels. The findings above offer fresh perspectives on how DNA methylation inhibitor reacts to different plant growth regulators. [ABSTRACT FROM AUTHOR]

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive. [ABSTRACT FROM AUTHOR]

Horizontal 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. In 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 LTR-retrotransposons from the Copia superfamily were transferred between these wild plant species, especially those of the Ivana and Ale lineages. This 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. Author summary: Horizontal transfer (HT) is known to occur in eukaryotes, but its prevalence among non-parasitic wild species is not well understood. Through genomic analysis of wild non-model species in the Massane forest, we identified 12 HT events primarily involving low copy number LTR-retrotransposons, with half occurring between lianas and trees, unveiling a novel route for HT between non-parasitic plants. [ABSTRACT FROM AUTHOR]

Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin® Soil from Macherey-Nagel, DNEasy® PowerSoil® from Qiagen, and E.Z.N.A.® Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge. [ABSTRACT FROM AUTHOR]

Foraging is one of the most fundamental activities contributing to the maximization of an animal's fitness, and thus herbivores must optimize their diet selection and intake to meet their nutrient demands for survival, growth, and reproduction. Using plant DNA barcoding, we determined diet composition of five subpopulations of adult female pronghorn antelope (Antilocapra americana) grazing rangelands in southern and southeastern Idaho, USA. Fecal samples were collected for two years (2018–2019), and across metabolically-important adult female life history stages (late gestation, early lactation, breeding season). Plant DNA barcoding yielded 137 detected species within pronghorn diets across subpopulations and sampling periods with forbs being the most abundant. Pronghorn dietary functional group composition ranged from 52.2–60.3% from forbs followed by shrubs (22.6–28.2%), graminoids (8.7–15.7%), and legumes (5.5–9.6%). Dietary protein intake was also highest from forbs and ranged from 32.4–62.4% followed by graminoids (1.2–43.1%), shrubs (18.7–21.3%), and legumes (2.6–7.4%). We found significant intra- and interannual differences in the mean number of genera-based plant detections in pronghorn diets. Dietary protein intake of cultivated legumes (e.g., alfalfa [Medicago sativa] and sainfoin [Onobrychis viciifolia]) was lower than expected, ranging from <1.0–30.8%, suggesting that even within an agricultural-dominated landscape, factors other than plant nutritional composition contributed to pronghorn diets. Although the plant DNA barcoding technique exhibits limitations, it demonstrated potential for elucidating pronghorn dietary species richness, particularly for plants consumed in small proportions, as well as for observing temporal fluctuations in functional group composition and dietary protein intake explained through the interplay between environmental factors, plant chemical composition, and the animals' physiological needs. [ABSTRACT FROM AUTHOR]

Background: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). Results: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. Conclusions: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork. [ABSTRACT FROM AUTHOR]

The aim of this work was to validate markers linked to the SH3 gene of coffee leaf rust (CLR) resistance and use them in assisted selection. Initially, we validated the markers in genotypes already known to carry SH3. Next, we performed phenotype and genotype evaluation for resistance to CLR in coffee plants growing under field conditions. We used Arabica coffee progenies derived from BA-10, which carries the SH3 gene due to introgression of C. liberica. Three SCAR markers (SP-M16-SH3, BA-48-21O-f, and BA-124-12K-f) and one SSR marker (Sat244) linked to SH3 gene were used to amplify the coffee plants' DNA. Our assessments of markers validation in resistant genotypes, SP-M16-SH3 and BA-124-12K-f, were efficient to identify the SH3 gene. These two markers were used to evaluate the progenies derived from BA-10 and were significantly linked to the phenotype evaluations. The SP-M16-SH3 marker was more efficient, with the advantage of being codominant. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) is a powerful conservation tool that has made significant advancements in the past decade. Since its initial application in 2008 to detect invasive tadpoles, eDNA has become popular for finding rare and invasive species that are otherwise difficult to detect through traditional field sampling methods. This study sought to determine a detectable range of eDNA for an aggressively invasive aquatic plant species, Hydrilla verticillata (L. f.) Royle, as well as to understand how stream discharge affects eDNA detection distance. To do so, we surveyed downstream of a large reservoir, which is separated from its distributary by a dam. Hydrilla verticillata is present in the reservoir but absent in the distributary. We used correlation analysis to understand the relationship between transportation distance and detection. We also built upon a model developed by Pont et al. (2018) to assess the relationships between stream discharge and eDNA detection distance, as well as to compare this relationship for our plant-based eDNA study with previous animal-based studies. By utilizing this natural system and surveying at different points downstream of the known population, this study detected H. verticillata eDNA nearly 5 km from the source population. This study, which is the 1st known instance of correlating plant eDNA to animal eDNA detection ranges, suggests the genetic material of both kingdoms are similarly detectable in flowing freshwaters and supports other studies that suggest discharge is a strong predictor of the detectable distance of eDNA. [ABSTRACT FROM AUTHOR]

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties. [ABSTRACT FROM AUTHOR]

Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9‐targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ (EVD) retrotransposon in wild‐type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage. Analysis of hemizygous T‐DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T‐DNA insertion mutants and transgenic plants. [ABSTRACT FROM AUTHOR]

With the development of urban industrialization, the increasing ozone concentration (O3) at ground level stresses on the survival of plants. Plants have to adapt to ozone stress. DNA methylation is crucial for a rapid response to abiotic stress in plants. Little information is known regarding the epigenetic response of DNA methylation of plants to O3 stress. This study is designed to explore the epigenetic mechanism and identify a possible core modification of DNA methylation or genes in the plant, in response to O3 stress. We investigated the agronomic traits and genome-wide DNA methylation variations of the Japonica rice cultivar Nipponbare in response to O3 stress at three high concentrations (80, 160, and 200 nmol·mol−1), simulated using open-top chambers (OTC). The flag leaf length, panicle length, and hundred-grain weight of rice showed beneficial effects at 80 nmol·mol−1 O3 and an inhibitory effect at both 160 and 200 nmol·mol−1 O3. The methylation-sensitive amplified polymorphism results showed that the O3-induced genome-wide methylation alterations account for 14.72–15.18% at three different concentrations. Our results demonstrated that methylation and demethylation alteration sites were activated throughout the O3 stress, mainly at CNG sites. By recovering and sequencing bands with methylation alteration, ten stress-related differentially amplified sequences, widely present on different chromosomes, were obtained. Our findings show that DNA methylation may be an active and rapid epigenetic response to ozone stress. These results can provide us with a theoretical basis and a reference to look for more hereditary information about the molecular mechanism of plant resistance to O3 pollution. [ABSTRACT FROM AUTHOR]

SUMMARY: Non‐homologous end joining (NHEJ) plays a major role in repairing DNA double‐strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals. Significance Statement: Non‐homologous end joining (NHEJ) is a major pathway to repair DNA double‐strand breaks and therefore is important for genome maintenance and editing. Using an evolutional analysis, we identified a plant ortholog of an NHEJ protein PAXX and contributed to understanding the exact molecular mechanism of NHEJ in plants. [ABSTRACT FROM AUTHOR]

DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2 µM for HPLC-UV and 0.02 µM for HPLC-MS of methylated cytosine. • New sensitive HPLC-UV method to estimate DNA methylation. • Even more sensitive one with HPLC–MS. • Method suitable for DNA isolates from fungi and plants. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants. [ABSTRACT FROM AUTHOR]

BACKGROUND: DNA fingerprinting is a Special technique used to determine the probable identity of a person based on the nucleotide sequences of certain regions of Plant DNA that are unique to individuals. AIMS AND OBJECTIVES: In this study, the random amplified polymorphic deoxyribonucleic acid (RAPD) technique was employed for the determination of the components in Ayurvedic herbal medicine. The RAPD approach is appealing to a large number of scientists since it may be implemented in a variety of settings with little effort. Most importantly, RAPD analysis does not require any cloning, sequencing, or other molecular characterization of the genome to extract a large number of genetic markers from a small amount of deoxyribonucleic acid (DNA). MATERIALS AND METHODS: Fresh leaves of the Thevetia peruviana (Pers.) Roxb plants were gathered in the month of June in the surroundings of Solapur. RESULTS: We used OPAB11, OPT12, OPQ15, and OPC104 decamer primers to conduct an RAPD study that measures genetic variation at random. Pure and RNA free high molecular weight DNA was extracted from the leaves. CONCLUSION: The DNA was amplified with repeat primers and 4 polymerase chain reaction primers, and then analyzed on agarose gels. Other wild T. peruviana species might be easier to track down with this method. [ABSTRACT FROM AUTHOR]

Background: Ziziphi spinosae semen (ZSS), dry mature seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F. Chow., are widely used in the treatment of insomnia, fright palpitations, and profuse dreaming in medical treatments. The counterfeit species of ZSS, Ziziphi mauritianae semen (ZMS), and Hovenia dulcis Thunb (HDT) are seeds from Ziziphus incurva Roxb. and Hovenia acerba Lindl., respectively. However, the pharmacopeia is not recorded for ZMS and HDT. In this article, we reviewed the identification methods of ZSS, ZMS, and HDT. Materials and Methods: The random amplified polymorphic DNA (RAPD) technique was used to identify the ZSS and its copies. Plant genome DNA was extracted from Z. jujuba Mill., Z. incurva Roxb., and H. acerba Lindl. Random primers were designed to amplify the genome. The amplified products were detected by agarose gel electrophoresis. Results: By analyzing the polymorphism of the amplified product DNA fragment, the DNA fingerprint maps of Z. jujuba Mill., Z. incurva Roxb., and H. acerba Lindl. were successfully constructed. [ABSTRACT FROM AUTHOR]

The periodical cicadas in the genus Magicicada are remarkable for their unusual life histories and dramatic synchronized emergences every 13 or 17 years. While aspects of their evolution, mating behaviors, and general biology have been well-characterized, there is surprising uncertainty surrounding the feeding habits of the short-lived adult stage. Despite a tentative scientific consensus to the contrary, the perception that adult Magicicada do not feed has persisted among the general public, and recent studies are lacking. We directly investigated the feeding behavior of Magicicada spp. through high-throughput sequencing (HTS)-based dietary analysis of nymphs, freshly molted (teneral) adults, and fully sclerotized adults collected from orchard and wooded habitats during the 2021 emergence of Brood X. Identifiable plant DNA (trnF , ITS amplicons) was successfully recovered from nymphs and adults. No plant DNA was recovered from teneral adults, suggesting that all DNA recovered from sclerotized adults was ingested during the post-teneral adult stage. Both nymphs and adults were found to have ingested a range of woody and herbaceous plants across 17 genera and 14 families. Significantly more plant genera per individual were recovered from adults than from nymphs, likely reflecting the greater mobility of the adult stage. We hypothesize that the demonstrated ingestion of plant sap by Magicicada adults is driven by a need to replace lost water and support specialized bacteriome-dwelling endosymbionts that cicadas depend upon for growth and development, which constitutes true feeding behavior. [ABSTRACT FROM AUTHOR]

[Display omitted] Preventing plant loss and improving their health status are essential for agricultural industry. Correspondingly, the deprivation of plants severely impacts our ecological system. As such, global efforts have been intensely made to promote the development of advanced sensing and treatment platforms to forestall plant loss. Existing technologies mainly encounter a number of challenges in providing results in a non-invasive, rapid turnaround, and affordable fashions. Accordingly, notable progressions in innovative approaches—particularly biosensing and delivery platforms, are vastly required for agriculture realm. In this regard, microneedles have emerged as a pivotal technological tool that plays multifaceted roles in biosensing and delivery systems, with attention of growing towards agriculture. Simply put, microneedles offer several advantages over conventional methods for being less invasive, rapid, and highly precise. In this review, recent advancements in microneedle technologies including their implementations in agriculture are highlighted coherently. In particular, extracting DNA from plant leaves and expressing transient genes using microneedles are elaborated in details. Microneedle-based sensing platforms for detecting essential compounds and secondary metabolites are discussed as well. Recent advances focusing the delivery of agrochemicals and nanotherapeutics via microneedles are elaborated. By this means, this review aims to bridge the existing gaps between microneedles and agriculture precisely. [ABSTRACT FROM AUTHOR]