Plant DNA Research Articles

Comparing DNA isolation methods for forest trees: quality, plastic footprint, and time-efficiency

BackgroundGenetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation.ResultsIn the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN.ConclusionsQIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.

Evolutionary trajectory of organelle-derived nuclear DNAs in the Triticum/Aegilops complex species.

Organelle-derived nuclear DNAs, nuclear plastid DNAs (NUPTs), and nuclear mitochondrial DNAs (NUMTs) have been identified in plants. Most, if not all, genes residing in NUPTs/NUMTs (NUPGs/NUMGs) are known to be inactivated and pseudogenized. However, the role of epigenetic control in silencing NUPGs/NUMGs and the dynamic evolution of NUPTs/NUMTs with respect to organismal phylogeny remain barely explored. Based on the available nuclear and organellar genomic resources of wheat (genus Triticum) and goat grass (genus Aegilops) within Triticum/Aegilops complex species, we investigated the evolutionary fates of NUPTs/NUMTs in terms of their epigenetic silencing and their dynamic occurrence rates in the nuclear diploid genomes and allopolyploid subgenomes. NUPTs and NUMTs possessed similar genomic atlas, including (i) predominantly located in intergenic regions and preferential integration to gene regulation regions and (ii) generating sequence variations in the nuclear genome. Unlike nuclear indigenous genes, the alien NUPGs/NUMGs were associated with repressive epigenetic signals, namely high levels of DNA methylation and low levels of active histone modifications. Phylogenomic analyses suggested that the species-specific and gradual accumulation of NUPTs/NUMTs accompanied the speciation processes. Moreover, based on further pan-genomic analyses, we found significant subgenomic asymmetry in the NUPT/NUMT occurrence, which accumulated during allopolyploid wheat evolution. Our findings provide insight into the dynamic evolutionary fates of organelle-derived nuclear DNA in plants.

Integrated widely targeted metabolomics and network pharmacology revealed quality disparities between Guizhou and conventional producing areas of Codonopsis Radix.

With the internationalization of traditional Chinese medicine, the demand for medicinal and edible Codonopsis Radix (CR) has increased, and its medicinal resources have attracted attention. CR is a well-known traditional Chinese medicine with a long pharmaceutical and edible history. The Guizhou province in China has abundant CR resources, but in the absence of systematic studies on species identification and chemical compositions, the capacity of the capacity of the province to CR resource has not been fully utilized. We used plant morphology and DNA barcoding techniques to identify Luodang (LD) and Weidang (WD) species. To investigate the differences in metabolites between LD and WD, as well as three Chinese Pharmacopeia CRs, and to predict pharmacological mechanisms of action for the dominant differential metabolites, we utilized widely targeted metabolomics and network pharmacology. The results also revealed the material basis for the excellent food properties of both LD and WD. The plant traits and DNA barcoding molecular identification results indicated that Luodang and Weidang from Guizhou were Codonopsis tangshen and Codonopsis pilosula, respectively. Widely targeted metabolomics analysis revealed that a total of 1,116 metabolites from 14 categories, including phenolic acids, lipids, flavonoids, were found in five CRs and shared 1,054 (94.4%) metabolites. LD and WD each contained 3 and 10 dominant differential metabolites, respectively, which were primarily flavonoids and amino acids. Amino acids, phenolic acids, and organic acids play important roles in their excellent food attributes. In CR, eight dominant differential metabolites were discovered for the first time, including isoorientin-7-O-(6″-feruloyl) glucoside, N-formyl-L-methionine, and cyclo (Phe-Glu), among others. Network pharmacology analyses showed that, in LD, dominant differential metabolites were closely related to anti-tumor, cardiovascular disease improvement, nervous system protection, and metabolic disease treatment, whereas in WD, they were closely related to nervous system protection and cardiovascular disease improvement. The species of LD and WD were included in the Chinese Pharmacopeia, and their metabolite profiles were remarkably similar to CR from traditional producing areas. Therefore, LD and WD can be used and promoted medicinally as CR, and they have potential value for new drug development. This study enriched the database of CR compounds and provided a reference for quality control, resource development, and new drug development of CR.

Targeted control of supporting pathways in paclitaxel biosynthesis with CRISPR-guided methylation.

Introduction: Plant cell culture biomanufacturing is rapidly becoming an effective strategy for production of high-value plant natural products, such as therapeutic proteins and small molecules, vaccine adjuvants, and nutraceuticals. Many of these plant natural products are synthesized from diverse molecular building blocks sourced from different metabolic pathways. Even so, engineering approaches for increasing plant natural product biosynthesis have typically focused on the core biosynthetic pathway rather than the supporting pathways. Methods: Here, we use both CRISPR-guided DNA methylation and chemical inhibitors to control flux through the phenylpropanoid pathway in Taxus chinensis, which contributes a phenylalanine derivative to the biosynthesis of paclitaxel (Taxol), a potent anticancer drug. To inhibit PAL, the first committed step in phenylpropanoid biosynthesis, we knocked down expression of PAL in Taxus chinensis plant cell cultures using a CRISPR-guided plant DNA methyltransferase (NtDRM). For chemical inhibition of downstream steps in the pathway, we treated Taxus chinensis plant cell cultures with piperonylic acid and caffeic acid, which inhibit the second and third committed steps in phenylpropanoid biosynthesis: cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL), respectively. Results: Knockdown of PAL through CRISPR-guided DNA methylation resulted in a profound 25-fold increase in paclitaxel accumulation. Further, through the synergistic action of both chemical inhibitors and precursor feeding of exogenous phenylalanine, we achieve a 3.5-fold increase in paclitaxel biosynthesis and a similar reduction in production of total flavonoids and phenolics. We also observed perturbations to both activity and expression of PAL, illustrating the complex transcriptional co-regulation of these first three pathway steps. Discussion: These results highlight the importance of controlling the metabolic flux of supporting pathways in natural product biosynthesis and pioneers CRISPR-guided methylation as an effective method for metabolic engineering in plant cell cultures. Ultimately, this work demonstrates a powerful method for rewiring plant cell culture systems into next-generation chassis for production of societally valuable compounds.

Physical Pretreatments Applied in Three Commercial Kits for the Extraction of High-Quality DNA from Activated Sewage Sludge.

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.

Seasonal shifts in pronghorn antelope (Antilocapra americana) diets under a new lens: Examining diet composition using a molecular technique.

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.

Edible flora in pre-Columbian Caribbean coprolites: Expected and unexpected data.

Coprolites, or mummified feces, are valuable sources of information on ancient cultures as they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vieques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and lifestyles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested during food consumption, to further support dietary habits. Our findings show that pre-Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.), papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton (Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phytopathogenic fungi and plant interactions confirmed the potential consumption of these plants as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucinogenic plants likely played an important role in ancient human subsistence and societal customs. We compared our results with coprolites found in Mexico and the United States, as well as present-day faeces from Mexico, Peru, and the United States. The results suggest that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers, while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-Columbian cultures and larger scale farmers and United States individuals. Our study highlights differences in dietary patterns related to human lifestyles and provides insight into the flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal specimens demonstrate the importance of ancient DNA studies to better understand pre-Columbian populations.

The two faces of DJ-1D proteins.

Despite the documented bi-enzymatic mode of methylglyoxal detoxification, the single-step catalysis of methylglyoxal by DJ-1/Pfp-I domain containing proteins has been in the limelight. Prasad et al. recently discovered another functional facet of these moonlighting proteins: the deglycase potential of DJ-1D to repair the glycated DNA, RNA, and proteins in plants.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

A review on Genetic markers with special reference to Genetic Diversity of Four Economically Important Trees of Myrtaceae Family of Braj Region, India

Callistemon viminalis (Sol. ex Gaertn.) Byrnes, Psidium guajava L., Syzygium cumini L. and Eucalyptus grandis W.Hill are members of the Myrtaceae family and are found growing in various locations throughout the Districts of Agra and Mathura. Although some trees can be found in tropically dry and xeric locations, these trees are found in tropical and subtropical regions. These plants have significant medical value, are frequently used customarily in the Braj region, and contain high amounts of secondary metabolites and polyphenols that prevent the separation of plant DNA. DNA isolation is a method for purifying DNA from plant elements by integrating various chemical and physical techniques. For the investigation of plant genetic variation, DNA markers like PCR (RAPD, SSLP, and AFLP) are often used methods that only need a minimal amount of DNA material. The most widely used method for examining genetic diversity is RAPD analysis. RAPD is utilized for a variety of tasks, including gene mapping, genetic identification, and investigations involving closely related species. This database review's main goal is to provide extensive, essential genetic information, methods, and applications. Significant connections between the history of a plant species and the makeup of its current population can be found thanks to genetic diversity. Researchers may utilize this information in the future to manage, cultivate, and improve the breeding programme for these plant species.

Base Excision DNA Repair in Plants: Arabidopsis and Beyond.

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.

Destruction of transgenic potato plants (Solanum tuberosum L.) remnants under the influence of soil microbiome

Scientific community has been considering various hypotheses regarding the possible impact of GM plants on non-target soil microorganisms for decades, distinguishing direct and indirect effects. Direct effects are usually easier to detect, as transgenic proteins, characterized by a range of elimination against certain types of insects, pathogenic fungi and bacteria, can affect non-target beneficial symbionts and soil microbial groups involved in the transformation of organic substances. Indirect effects are difficult to assess, as many different factors can affect the composition of root exudates and the metabolic activity of plants. Changes in metabolic pathways can affect the composition of soil exudates and gene expression in plant tissues. At the same time, these changes can affect the decomposition of organic matter and the course of microbiological processes in the soil. Based on experimental studies, an assessment of the rate of destruction of plant residues and DNA degradation in transgenic potato plants in soddy-podzolic soil of potato crop rotation was carried out. The results of the studies have established a significant role of the microbiome of soddy-podzolic soil of potato crop rotation in the destruction of residues of transgenic potato plants and DNA degradation. Destruction of residues of transgenic plants under the influence of microflora of soddy-podzolic soil of potato crop rotation was 30 days with a loss of raw mass 87.14±1.11%. DNA degradation over this period is 88% (186 ng/mg) from the initial amount of total DNA, and degradation of nptII and 35S genes — about 90% from their initial amount in PCR products. It was found that about 90% of nptII and 35S genes in DNA residues of transgenic potato plants under the influence of microbiome soddy-podzolic soil occurs on day 30.

Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay

Rice false smut, caused by Ustilaginoidea virens, is a devastating disease that greatly reduces rice yield and quality. However, controlling rice false smut disease is challenging due to the unique infection mode of U. virens. Therefore, there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease. Lateral flow strip-based recombinase polymerase amplification (LF-RPA) overcomes the limitations of current U. virens detection technologies, which are time-consuming, require delicate equipment, and have a high false-positive rate. In this study, we used a comparative genomics approach to identify Uv_3611, a specific gene of U. virens, as the target for the LF-RPA assay. The designed primers and probe efffectively detected the genomic DNA (gDNA) of U. virens and demonstrated no cross-reactivity with related pathogens. Under optimal conditions, the LF-RPA assay demonstrated a sensitivity of 10 pg of U. virens gDNA. Additionally, by incorporating a simplified PEG-NaOH method for plant DNA extraction, the LF-RPA assay enabled the detection of U. virens in rice spikelets within 30 min, without the need for specialized equipment. Furthermore, the LF-RPA assay successfully detected U. virens in naturally infected rice and seed samples in the field. Therefore, the LF-RPA assay is sensitive, efficient, and convenient, and could be developed as a kit for monitoring rice false smut disease in the field.

Evaluation of the Genetic Stability of Amaranthus viridis L. Species in Selected Regions of Western Ghats using Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA is cheap and fast molecular technique to identify the genetic resemblances or variations in DNA in various plants. The proposed research work wasintendedto compare the genetic diversification of Amaranthus viridis species in selected regions of Western Ghats in Tamilnadu and Kerala by RAPD analysis.The biometric morphological data among the five A. viridis accessions were compared. Their genomic DNA was isolated and finger prints were obtained using three RAPD markers. The percentage polymorphism, polymorphism information content, effective multiplex ration and resolving power were calculated. UPGMA dendrogram was constructed and their genetic relatedness was compared using Jaccard coefficient.Upon finger printing, 66 bands were counted for the three RAPD primers used,among which 46 bands were polymorphic band numbers from 8 to 21. Percentage of polymorphic bands ranged between 44.44% and 80.77%. EMRvalues for 66 polymorphic loci ranged between 3.56 and 17.01. The UPGMA dendrogram comparing the genomic profiles of the 5 A. viridis accessions using Jaccard coefficient revealed their genetic distances where the maximum Jaccard’s coefficient value observed was 0.96 between AVKATN and AVNEK. Among the 5 selections the peak similarity index (0.9565) was witnessed between AVKATN and AVNEK. The cuurent study revealed that AVKATN and AVNEK had the highest their genetic distances, whereas AVKATN and AVNEK were genetically similar among the 5 A. viridis accessions. Several polymorphic bands which ranged up to 80.77% have proved their genetic variations.

ADVANCEMENT OF CROP PRODUCTIVITY VIA CRISPR-NANOPARTICLE INTERFACE

Plant improvement strategies involve diverse techniques, ranging from traditional to marker-assisted methods, as well as chemical and radiation treatments. However, these methods can introduce imprecise changes in plant DNA. Accelerating plant enhancement is crucial to meet global food demand, but current methods are time-consuming. Scientists are revolutionizing plant breeding by employing various techniques to develop crops with specific attributes, such as increased yield and pest resistance, aligning with environmental and societal needs. While these methods offer substantial advantages, they often face challenges and can be less precise than desired. Innovative methods, such as gene editing using CRISPR, offer enhanced precision. CRISPR technology enables precise modifications to a plant's DNA, allowing for targeted improvements without unintended consequences. While CRISPR shows great potential, ensuring its safe and accurate implementation is a priority. Scientists are exploring diverse methods, both viral and non-viral, to effectively deliver CRISPR components into plant cells, with non-viral approaches gaining traction due to their safety and versatility. Nanoparticles play a pivotal role in these advancements by serving as delivery vehicles for CRISPR tools. These particles safeguard and transport the necessary components to specific locations within plants, bolstering growth, yield, and disease resistance. Despite challenges, the synergy of nanotechnology and CRISPR holds promise for revolutionizing plant improvement while safeguarding the environment. This integrated approach offers the potential to enhance crop growth and quality while upholding ecological balance.

Investigation of the Influence of Polyamines on Mature Embryo Culture and DNA Methylation of Wheat (Triticum aestivum L.) Using the Machine Learning Algorithm Method.

Numerous factors can impact the efficiency of callus formation and in vitro regeneration in wheat cultures through the introduction of exogenous polyamines (PAs). The present study aimed to investigate in vitro plant regeneration and DNA methylation patterns utilizing the inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) methods in wheat. This investigation involved the application of distinct types of PAs (Put: putrescine, Spd: spermidine, and Spm: spermine) at varying concentrations (0, 0.5, 1, and 1.5 mM). The subsequent outcomes were subjected to predictive modeling using diverse machine learning (ML) algorithms. Based on the specific polyamine type and concentration utilized, the results indicated that 1 mM Put and Spd were the most favorable PAs for supporting endosperm-associated mature embryos. Employing an epigenetic approach, Put at concentrations of 0.5 and 1.5 mM exhibited the highest levels of genomic template stability (GTS) (73.9%). Elevated Spd levels correlated with DNA hypermethylation while reduced Spm levels were linked to DNA hypomethylation. The in vitro and epigenetic characteristics were predicted using ML techniques such as the support vector machine (SVM), extreme gradient boosting (XGBoost), and random forest (RF) models. These models were employed to establish relationships between input variables (PAs, concentration, GTS rates, Msp I polymorphism, and Hpa II polymorphism) and output parameters (in vitro measurements). This comparative analysis aimed to evaluate the performance of the models and interpret the generated data. The outcomes demonstrated that the XGBoost method exhibited the highest performance scores for callus induction (CI%), regeneration efficiency (RE), and the number of plantlets (NP), with R2 scores explaining 38.3%, 73.8%, and 85.3% of the variances, respectively. Additionally, the RF algorithm explained 41.5% of the total variance and showcased superior efficacy in terms of embryogenic callus induction (ECI%). Furthermore, the SVM model, which provided the most robust statistics for responding embryogenic calluses (RECs%), yielded an R2 value of 84.1%, signifying its ability to account for a substantial portion of the total variance present in the data. In summary, this study exemplifies the application of diverse ML models to the cultivation of mature wheat embryos in the presence of various exogenous PAs and concentrations. Additionally, it explores the impact of polymorphic variations in the CRED-iPBS profile and DNA methylation on epigenetic changes, thereby contributing to a comprehensive understanding of these regulatory mechanisms.

Do adult Magicicada (Hemiptera: Cicadidae) feed? Historical perspectives and evidence from molecular gut content analysis.

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.

The aphid Myzus persicae (Hemiptera: Aphididae) acquires chloroplast DNA during feeding on host plants.

Aphids (Hemiptera: Aphididae) extract nutrients from host plant phloem via stylets that facilitate salivation and sap uptake. When navigating to the phloem, aphids periodically puncture nonvascular cells and sample cell contents, but rarely cause significant cell damage. As a result, aphids are considered "stealthy" feeders. In contrast, insects that do cause damage, such as chewing herbivores, will take up host cell contents-including DNA-into their guts. Researchers can use molecular barcoding methods to identify recent host use patterns of chewing herbivores. This information is valuable for both pest management and basic ecological studies. Because of their stealthy feeding style, it was assumed that host plant DNA could not be recovered from aphids and other Sternorrhyncha. However, several recent studies document host plant DNA uptake by psyllids, which feed in a similar manner to aphids. Therefore, we hypothesized that aphids may also acquire DNA from host plants. Since aphids puncture and sample cytosol contents from cells, we predicted that aphids would be most likely to acquire DNA from chloroplasts. To test this, we performed host feeding and host transfer experiments with Myzus persicae (Sulzer), then used PCR to recover and sequence a region between the trnT and trnF genes from acquired chloroplast DNA. We found that M. persicae readily acquires chloroplast DNA, even prior to phloem contact, and that fragment sizes sufficient for host plant identification can be recovered. Our work suggests that molecular gut content analysis is a viable tool for studying aphid-host interactions.

Rapid detection and quantification of Aphanomyces cochlioides in sugar beet

Sugar beet (Beta vulgaris L.) is an economically important crop in temperate climates providing nearly 30% of sugar production worldwide. The oomycete Aphanomyces cochlioides is the causative agent of seedling damping-off and root rot disease in sugar beet. The pathogen is responsible for plant degeneration and drastic yield losses in all major sugar beet producing areas. The identification of resistant germplasm is essential to reduce the use of chemical treatments as well as the costs of protective measures and to effectively limit the damage caused by the pathogen. In this study we aimed to establish a qPCR-based method to quantify the pathogen DNA in infected plants and to predict the resistance levels of different sugar beet genotypes in response to A. cochlioides. The difference in the response to A. cochlioides isolates with different geographical origins was investigated. In addition, confocal microscopy was performed in order to observe the spatial and temporal colonization pattern in infected seedlings of susceptible and partially resistant breeding lines. The research presented in this article provides a tool to understand the progress of the infection in infected tissues and to identify the genetic background of resistance to A. cochlioides that can be used to support breeding programs.

Browsers or Grazers? New Insights into Feral Burro Diet Using a Non-Invasive Sampling and Plant DNA Metabarcoding Approach.

Ungulates play a large role in shaping ecosystems and communities by influencing plant composition, structure, and productivity. We investigated the summer diets of feral burros in two ecosystems in which they are found in the United States: a subtropical desert in Arizona and a temperate juniper shrubland in Utah. Between 24 June and 16 July of 2019, we gathered 50 burro fecal samples from each location and used plant DNA metabarcoding to determine the burros' diets. We found that during our sampling period the burros in the Sonoran Desert consumed a higher proportion of woody browse and had a narrower dietary niche breadth and lower degree of diet diversity compared to the burros in the juniper shrubland ecosystem, where the burros consumed higher proportions of graminoids and forbs and had a higher diet diversity index and broader dietary niche breadth. The burros in the Sonoran Desert relied primarily on Prosopis spp. (mesquite) and Poaceae grasses, whereas the burros in the juniper shrubland relied on a wider variety of forb and grass species, likely due to the greater variability in the forage species temporally and spatially available in that temperate ecosystem. We found that feral burros are highly adaptable with respect to diet and appear to be employing a mixed feeding strategy, similar to their ancestor, the African wild ass, to meet their nutritional needs in whichever ecosystem they are found.