Sample and Library Preparation for PacBio Long-Read Sequencing in Grapevine.

PacBio long-read sequencing is a third-generation technology that generates long reads up to 20 kilobases (kb), unlike short-read sequencing instruments that produce up to 600 bases. Long-read sequencing is particularly advantageous in higher organisms, such as humans and plants, where repetitive regions in the genome are more abundant. The PacBio long-read sequencing uses a single molecule, real-time approach where the SMRT cells contain several zero-mode waveguides (ZMWs). Each ZMW contains a single DNA molecule bound by a DNA polymerase. All ZMWs are flushed with deoxy nucleotides with a fluorophore specific to each nucleotide. As the sequencing proceeds, the detector detects the wavelength of the fluorescence and the nucleotides are read in real-time. This chapter describes the sample and library preparation for PacBio long-read sequencing for grapevine.

[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

Long-read sequencing of extrachromosomal circular DNA and genome assembly of a Solanum lycopersicum breeding line revealed active LTR retrotransposons originating from S. Peruvianum L. introgressions.

Transposable elements (TEs) are a major force in the evolution of plant genomes. Differences in the transposition activities and landscapes of TEs can vary substantially, even in closely related species. Interspecific hybridization, a widely employed technique in tomato breeding, results in the creation of novel combinations of TEs from distinct species. The implications of this process for TE transposition activity have not been studied in modern cultivars. In this study, we used nanopore sequencing of extrachromosomal circular DNA (eccDNA) and identified two highly active Ty1/Copia LTR retrotransposon families of tomato (Solanum lycopersicum), called Salsa and Ketchup. Elements of these families produce thousands of eccDNAs under controlled conditions and epigenetic stress. EccDNA sequence analysis revealed that the major parts of eccDNA produced by Ketchup and Salsa exhibited low similarity to the S. lycopersicum genomic sequence. To trace the origin of these TEs, whole-genome nanopore sequencing and de novo genome assembly were performed. We found that these TEs occurred in a tomato breeding line via interspecific introgression from S. peruvianum. Our findings collectively show that interspecific introgressions can contribute to both genetic and phenotypic diversity not only by introducing novel genetic variants, but also by importing active transposable elements from other species.

Conserved DNA sequence analysis reveals the phylogeography and evolutionary events of Akebia trifoliata in the region across the eastern edge of the Tibetan Plateau and subtropical China.

BACKGROUND: The eastern edge of the Qinghai‒Tibet Plateau (QTP) and subtropical China have various regions where plant species originate and thrive, but these regions have been the focus of very few integrative studies. Here, we elucidated the phylogeographic structure of a continuous and widespread Akebia trifoliata population across these two regions. RESULTS: Sixty-one populations consisting of 391 genotypes were examined to assess population diversity and structure via network distribution analysis, maximum likelihood phylogenetic tree reconstruction, divergence time estimation, demographic history inference, and ancestral area reconstruction of both conserved internal transcribed spacer (ITS) and chloroplast (rps16) DNA sequences. The results showed that the ITS region was more variable than the rps16 region and could be suitable for studying intraspecific phylogeography. The A. trifoliata population displayed high genetic diversity, genetic differentiation and obvious phylogeographical structure, possibly originating on the eastern QTP, expanding during the last glacial-interglacial cycle, diverging in the early Pleistocene and middle Pleistocene, and extensively migrating thereafter. The migration route from west to east along rivers could be largely responsible for the long-distance dispersal of this species, while three main refuges (Qinba Mountains, Nanling Mountains and Yunnan-Guizhou Plateau) with multiple ice shelters facilitated its wide distribution. CONCLUSIONS: Our results suggested that the from west to east long migration accompanying with the minor short reciprocal migration in the south-north direction, and the three main refuges (the Qinba Mountains, Nanling Mountains and Yunnan-Guizhou Plateau) contributed to the extant geographical distribution of A. trifoliata. In addition, this finding also strongly reduced the discrepancy between glacial contraction and postglacial expansion and the in situ survival hypothesis by simultaneously considering the existence of many similar climate-related ecological niches and migration influences.

[Genetic background of lily germplasm resources based on SSR markers].

To study the genetic background of lily (Lilium spp.) germplasm resources, and accurately evaluate and select excellent germplasm for genetic improvement of lily, we analyzed the genetic background of 62 lily germplasm accessions from 11 provinces of China by using simple sequence repeat (SSR) molecular markers. The results showed that 15 out of 83 pairs of lily SSR primers were polymorphic. A total of 157 allelic loci were amplified, with the number of alleles per locus ranging from 5 to 19 and the average number of effective alleles per locus being 4.162 8. The average observed heterozygosity and expected heterozygosity were 0.228 2 and 0.694 1, respectively. The average polymorphic information content was 0.678 8. The average Nei's diversity index and Shannon's information index were 0.694 1 and 1.594 9, respectively, indicating that the tested lily germplasm had high genetic diversity. The 62 germplasm accessions were classified into 5 groups by the unweighted pair group method with arithmetic mean (UPGMA) and into 3 groups by the principal component analysis. The two analyses revealed a geographic correlation among different groups. The majority of lily germplasm accessions from the same source tended to cluster together. The population structure analysis classified the lily accessions into 4 populations and 1 mixed population. The above results provide a theoretical basis and genetic resources for the precise identification and breeding of lily germplasm resources.

Identification of AtALKBH1A and AtALKBH1D as DNA N6-adenine demethylases in Arabidopsis thaliana.

DNA N6-methyladenine (6 mA) has recently been discovered as a novel DNA modification in animals and plants. In mammals, AlkB homolog 1 (ALKBH1) has been identified as a DNA 6 mA demethylase. ALKBH1 tightly controls the DNA 6 mA methylation level of mammalian genomes and plays important role in regulating gene expression. DNA 6 mA methylation has also been reported to exist in plant genomes, however, the plant DNA 6 mA demethylases and their function remain largely unknown. Here we identify homologs of ALKBH1 as DNA 6 mA demethylases in Arabidopsis. We discover that there are four homologs of ALKBH1, AtALKBH1A, AtALKBH1B, AtALKBH1C and AtALKBH1D, in Arabidopsis. In vitro enzymatic activity studies reveal that AtALKBH1A and 1D can efficiently erase DNA 6 mA methylation. Loss of function of AtALKBH1A and AtALKBH1D causes elevated DNA 6 mA methylation levels in vivo. atalkbh1a/1d mutant displays delayed seed gemination. Based on our RNA-seq data, we find some regulators of seed gemination are dysregulated in atalkbh1a/1d, and the dysregulation is correlated with changes of DNA 6 mA methylation levels. This study identifies plant DNA 6 mA demethylases and reports the function of DNA 6 mA methylation in regulating seed germination.

Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA.

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.

Population epigenetics: DNA methylation in the plant omics era.

DNA methylation plays an important role in many biological processes. The mechanisms underlying the establishment and maintenance of DNA methylation are well understood thanks to decades of research using DNA methylation mutants, primarily in Arabidopsis (Arabidopsis thaliana) accession Col-0. Recent genome-wide association studies (GWASs) using the methylomes of natural accessions have uncovered a complex and distinct genetic basis of variation in DNA methylation at the population level. Sequencing following bisulfite treatment has served as an excellent method for quantifying DNA methylation. Unlike studies focusing on specific accessions with reference genomes, population-scale methylome research often requires an additional round of sequencing beyond obtaining genome assemblies or genetic variations from whole-genome sequencing data, which can be cost prohibitive. Here, we provide an overview of recently developed bisulfite-free methods for quantifying methylation and cost-effective approaches for the simultaneous detection of genetic and epigenetic information. We also discuss the plasticity of DNA methylation in a specific Arabidopsis accession, the contribution of DNA methylation to plant adaptation, and the genetic determinants of variation in DNA methylation in natural populations. The recently developed technology and knowledge will greatly benefit future studies in population epigenomes.

Molecular basis of chromatin remodelling by DDM1 involved in plant DNA methylation.

Eukaryotic gene regulation occurs at the chromatin level, which requires changing the chromatin structure by a group of ATP-dependent DNA translocases-namely, the chromatin remodellers1. In plants, chromatin remodellers function in various biological processes and possess both conserved and plant-specific components2-5. DECREASE IN DNA METHYLATION 1 (DDM1) is a plant chromatin remodeller that plays a key role in the maintenance DNA methylation6-11. Here we determined the structures of Arabidopsis DDM1 in complex with nucleosome in ADP-BeFx-bound, ADP-bound and nucleotide-free conformations. We show that DDM1 specifically recognizes the H4 tail and nucleosomal DNA. The conformational differences between ADP-BeFx-bound, ADP-bound and nucleotide-free DDM1 suggest a chromatin remodelling cycle coupled to ATP binding, hydrolysis and ADP release. This, in turn, triggers conformational changes in the DDM1-bound nucleosomal DNA, which alters the nucleosome structure and promotes DNA sliding. Together, our data reveal the molecular basis of chromatin remodelling by DDM1.

DNA Barcoding and Fertilization Strategies in Sideritis syriaca subsp. syriaca, a Local Endemic Plant of Crete with High Medicinal Value.

Herein, we applied DNA barcoding for the genetic characterization of Sideritis syriaca subsp. syriaca (Lamiaceae; threatened local Cretan endemic plant) using seven molecular markers of cpDNA. Five fertilization schemes were evaluated comparatively in a pilot cultivation in Crete. Conventional inorganic fertilizers (ChFs), integrated nutrient management (INM) fertilizers, and two biostimulants were utilized (foliar and soil application). Plant growth, leaf chlorophyll fluorescence, and color were assessed and leaf content of chlorophyll, key antioxidants (carotenoids, flavonoids, phenols), and nutrients were evaluated. Fertilization schemes induced distinct differences in leaf shape, altering quality characteristics. INM-foliar and ChF-soil application promoted yield, without affecting tissue water content or biomass partitioning to inflorescences. ChF-foliar application was the most stimulatory treatment when the primary target was enhanced antioxidant contents while INM-biostimulant was the least effective one. However, when the primary target is yield, INM, especially by foliar application, and ChF, by soil application, ought to be employed. New DNA sequence datasets for the plastid regions of petB/petD, rpoC1, psbK-psbI, and atpF/atpH were deposited in the GenBank for S. syriaca subsp. syriaca while the molecular markers rbcL, trnL/trnF, and psbA/trnH were compared to those of another 15 Sideritis species retrieved from the GenBank, constructing a phylogenetic tree to show their genetic relatedness.

Negative plant-soil feedback in Arabidopsis thaliana: Disentangling the effects of soil chemistry, microbiome, and extracellular self-DNA.

Nutrient deficiency, natural enemies and litter autotoxicity have been proposed as possible mechanisms to explain species-specific negative plant-soil feedback (PSF). Another potential contributor to negative PSF is the plant released extracellular self-DNA during litter decay. In this study, we sought to comprehensively investigate these hypotheses by using Arabidopsis thaliana (L.) Heynh as a model plant in a feedback experiment. The experiment comprised a conditioning phase and a response phase in which the conditioned soils underwent four treatments: (i) addition of activated carbon, (ii) washing with tap water, (iii) sterilization by autoclaving, and (iv) control without any treatment. We evaluated soil chemical properties, microbiota by shotgun sequencing and the amount of A. thaliana extracellular DNA in the differently treated soils. Our results showed that washing and sterilization treatments mitigated the negative PSF effect. While shifts in soil chemical properties were not pronounced, significant changes in soil microbiota were observed, especially after sterilization. Notably, plant biomass was inversely associated with the content of plant self-DNA in the soil. Our results suggest that the negative PSF observed in the conditioned soil was associated to increased amounts of soilborne pathogens and plant self-DNA. However, fungal pathogens were not limited to negative conditions, butalso found in soils enhancing A.thaliana growth. In-depth multivariate analysis highlights that the hypothesis of negative PSF driven solely by pathogens lacks consistency. Instead, we propose a multifactorial explanation for the negative PSF buildup, in which the accumulation of self-DNA weakens the plant's root system, making it more susceptible to pathogens.

DNA barcoding of Notopterygii Rhizoma et Radix (Qiang-huo) and identification of adulteration in its medicinal services.

Safety concerns, stemming from the presence of complex and unpredictable adulterants, permeate the entire industrial chain of traditional Chinese medicines (TCMs). The Notopterygii Rhizoma et Radix (NReR) from the Apiaceae family, commonly known as "Qiang-huo", is a widely used herbal medicine. The recent surge in its demand has given rise to a proliferation of counterfeit and substituted products in the market. Traditional identification presents inherent limitations, while DNA mini-barcoding, reliant on sequencing a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In this study, we constructed a comprehensive Internal Transcribed Spacer 2 (ITS2) matrix encompassing genuine NReR and their commonly found adulterants for the first time. Leveraging this matrix, we conducted a thorough assessment of the genetic profiles and sources of NReR available in the Chinese herbal medicine market. Following established DNA barcoding protocols, the intra-specific genetic divergences within NReR species were found to be lower than the inter-specific genetic divergences from other species. Among the 120 samples that were successfully amplified, ITS2 exhibits an outstanding species-level identification efficiency of 100% when evaluated using both the BLASTN and neighbor-joining (NJ) tree methods. We concluded that ITS2 is a mini-barcode that has shown its potential and may become a universal mini-barcode for the quality control of "Qiang-huo", thereby ensuring the safety of clinical medication.

Morpho-anatomical characterization and DNA barcoding of Artemesia vulgaris L.

Artemisia vulgaris L. belongs to Asteraceae, is a herbal plant that has various benefits in the medical field, so that its use in the medical field can be explored optimally, the plant must be thoroughly identified. This study aims to identify A. vulgaris both in terms of descriptive morpho-anatomy and DNA barcoding using BLAST and phylogenetic tree reconstruction. The morpho-anatomical character was observed on root, stem, and leaf. DNA barcoding analysis was carried out through amplification and alignment of the rbcL and matK genes. All studies were conducted on three samples from Taman Husada (Medicinal Plant Garden) Graha Famili Surabaya, Indonesia. The anatomical slide was prepared by the paraffin method. Morphological studies revealed that the leaves of A. vulgaris both on the lower-middle part and on the upper part of the stem have differences, especially in the character of the stipules, petioles, and incisions they have. Meanwhile, from the study of anatomy, A. vulgaris has an anomocytic type of stomata and its distribution is mostly on the ventral part of the leaves. Through the BLAST process and phylogenetic tree reconstruction, the plant sequences being studied are closely related to several species of the genus Artemisia as indicated by a percentage identity above 98% and branch proximity between taxa in the reconstructed phylogenetic tree.

First nanoplate digital PCR method to trace allergenic foods: Improved sensitivity for the detection of sesame.

Sesame (Sesamum indicum L.) is an important allergenic food whose presence can be the cause of severe allergic reactions in sensitised individuals. In this work, nanoplate digital PCR (ndPCR) was used to develop two methods to detect trace amounts of sesame in processed foods and compared with previously proposed real-time PCR assays. Two independent ndPCR approaches were successfully advanced, achieving sensitivities of 5 and 0.1 mg/kg of sesame in dough/biscuits, targeting the CO6b-1 and ITS regions, respectively. The sensitivity using both targets was improved by one order of magnitude comparing with real-time PCR and was not affected by food processing. CO6b-1 system was not influenced by food matrix, exhibiting similar performance regardless the use of complex matrix extracts or serial diluted DNA. Herein, ndPCR was proposed for the first time for the detection of allergenic foods with the advantage of providing better performance than real-time PCR regarding sensitivity and robustness.

First report of Ageratum yellow vein virus infecting papaya in Lampung, Indonesia.

BACKGROUND: Papaya (Carica papaya) is a tropical fruit of great economic and nutritional importance, loved for its sweet and delicious flesh. However, papaya cultivation faces serious challenges in the form of Begomovirus attacks. Begomoviruses are a group of viruses that pose a serious threat to plants worldwide. Including papaya, Begomovirus has become a significant threat to papaya production in various parts of the world and has been identified in several regions in Indonesia. METHODS: DNA was extracted from seven samples representing different papaya growing areas using a Plant Genomic DNA Mini Kit. Genomic DNA from the samples was subjected to PCR using universal primers of AC2, AC1, SPG1 and SPG2. The PCR products then sequenced using the dideoxy (Sanger) approach. The obtained sequence then compared to the gene bank using BLAST software available at NCBI. Multiple sequence alignment and phylogenetic tree construction were analyzed using the MEGA11 program. RESULTS: Detection based on viral nucleic acid in papaya plants in Pesawaran, Lampung Province with seven sampling points using universal primers SPG1/SPG2 showed positive results for Begomovirus infection with visible DNA bands measuring ± 900 bp. Direct nucleotide sequencing using SPG1/SPG2 primers for the AC2 and AC1 genes of the Begomovirus and confirmed by the BLAST program showed that papaya samples were infected with Ageratum yellow vein virus (AYVV). The phylogenetic results show that AYVV from papaya samples has a close relationship with the AYVV group from several other countries, with 98% homology. CONCLUSION: In the papaya cultivation area in Pesawaran, Lampung province, it was identified as Begomovirus, Ageratum yellow vein virus (AYVV) species and is closely related to the AYVV group from several other countries. Overall, our study further suggests that Ageratum acts as an alternative host and reservoir for Begomovirus.

Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding.

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

Spatial and temporal characterization of the rich fraction of plastid DNA present in the nuclear genome of Moringa oleifera reveals unanticipated complexity in NUPTs´ formation.

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.

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

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.

Plant-derived environmental DNA complements diversity estimates from traditional arthropod monitoring methods but outperforms them detecting plant-arthropod interactions.

Our limited knowledge about the ecological drivers of global arthropod decline highlights the urgent need for more effective biodiversity monitoring approaches. Monitoring of arthropods is commonly performed using passive trapping devices, which reliably recover diverse communities, but provide little ecological information on the sampled taxa. Especially the manifold interactions of arthropods with plants are barely understood. A promising strategy to overcome this shortfall is environmental DNA (eDNA) metabarcoding from plant material on which arthropods leave DNA traces through direct or indirect interactions. However, the accuracy of this approach has not been sufficiently tested. In four experiments, we exhaustively test the comparative performance of plant-derived eDNA from surface washes of plants and homogenized plant material against traditional monitoring approaches. We show that the recovered communities of plant-derived eDNA and traditional approaches only partly overlap, with eDNA recovering various additional taxa. This suggests eDNA as a useful complementary tool to traditional monitoring. Despite the differences in recovered taxa, estimates of community α- and ß-diversity between both approaches are well correlated, highlighting the utility of eDNA as a broad scale tool for community monitoring. Last, eDNA outperforms traditional approaches in the recovery of plant-specific arthropod communities. Unlike traditional monitoring, eDNA revealed fine-scale community differentiation between individual plants and even within plant compartments. Especially specialized herbivores are better recovered with eDNA. Our results highlight the value of plant-derived eDNA analysis for large-scale biodiversity assessments that include information about community-level interactions.

Identifying a suspect powder as a cannabis concentrate through chemical analysis and DNA testing.

PURPOSE: Cannabis is regulated in many countries, and cannabis products are diversifying, which can hinder identification. Here, we report the seizure of a powder sample with a cannabis-like odor in a spice bottle labeled "nutmeg" and identification of the sample by chemical testing and cannabis DNA testing. METHODS: The sample was observed under a microscope, extracted with methanol, and analyzed by gas chromatography-mass spectrometry (GC-MS). The chemical profile of the seized powder was compared with that of nutmeg samples. Gas chromatography-flame ionization detection was used to estimate the total Δ9-tetrahydrocannabinol (Δ9-THC) concentration in the sample. A commercially available cannabis DNA testing kit was used to confirm the presence of cannabis plant DNA in the seized sample. RESULTS: The characteristics of cannabis in the seized powder were difficult to determine through microscopic observation alone. GC-MS analysis identified ß-caryophyllene (an aromatic component of cannabis) and five cannabinoids unique to cannabis, including Δ9-THC. No common compounds were identified in the seized powder or nutmeg samples. The total Δ9-THC concentration in the sample was very high (approximately 47% by weight). Cannabis DNA testing confirmed that the seized powder contained cannabis. CONCLUSIONS: The seized powder was found to be a processed product made from a finely pulverized resin-like cannabis concentrate. Our results indicate that combined chemical and DNA analysis should help identify cannabis-related samples in various forms.