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Background: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus, we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of QC of genetic testing for processed foods., Objective: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance., Methods: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests between laboratories., Results: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability between laboratories., Conclusion: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for QC of genetic testing but also for food testing, medical diagnostics, and other applications in a range of fields., Highlights: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The number of food products with genetically modified (GM) crops on the global market has increased due to advancements in genetic engineering technology. Legislation regulating the labeling and use of GM crops has increased considerably worldwide to provide consumers with health and safety assurance. It is still unclear whether genetically modified organisms (GMOs) are present in the food market of the Kingdom of Saudi Arabia due to a lack of scientific studies. This work was planned to detect GM rice and GM potatoes in the Saudi food market. One hundred non-labeled rice and rice product samples and 50 potato and potato samples were collected randomly from different market sites of Makkah, Riyadh and Jeddah during 2022-2023. The cetyl trimethyl ammonium bromide (CTAB) method was used to extract DNA. Viviants DNA extraction kit was used to extract DNA from rice starch and potato chips. To find GMOs in samples, CMOScreen 35S and NOS test kits were utilized. DNA-based qualitative and quantitative approaches were used to screen targets for PCR detection of GM rice sequences. The results indicated that 32 (32%) rice samples were positive for CaMV 35S promoter, while no positive result was detected for the NOS terminator. Besides, 30% of potato samples were positive for the CaMV 35S promoter, and the same samples were positive for the presence of the Cry V gene. It could be concluded that there were GM rice and potatoes in the Kingdom of Saudi Arabia's food markets. Establishing strong regulations and certified laboratories to monitor genetically modified foods (GMF) or crops in the Saudi market is recommended.

This work reports the development and application of a disposable amperometric sensor built on magnetic microcarriers coupled to an Express PCR strategy to amplify a specific DNA fragment of the chloroplast trnH-psbA. The procedure involves the selective capture of a 68-mer synthetic target DNA (or unmodified PCR products) through sandwich hybridization with RNA capture probe-modified streptavidin MBs and RNA signaling probes, labeled using antibodies specific to the heteroduplexes and secondary antibodies tagged with horseradish peroxidase. Amperometric measurements were performed on screen-printed electrodes using the H 2 O 2 /hydroquinone system. Achieving a LOD of 3 pM for the synthetic target, it was possible to detect 2.5 pg of peanut DNA and around 10 mg kg -1 of peanut in binary mixtures (defatted peanut flours prepared in spelt wheat). However, the detectability decreased between 10 and 1000 times in processed samples depending on the treatment. The Express PCR-bioplatform was applied to the detection of peanut traces in foodstuff., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n = 147)., (© 2024 Wiley‐VCH GmbH.)

Since the establishment of procedures for the safety assessment of food products that use recombinant DNA technology, the manufacture, import, and sale of genetically modified (GM) foods that have not undergone safety assessment are prohibited under the Food Sanitation Act. Therefore, a performance study to confirm the GM food testing operations of each laboratory is very important to ensure the reliability of the GM food monitoring system. In 2022, GM papaya line PRSV-YK-which has not yet been authorized in Japan-was selected for testing, and a papaya paste and a DNA solution were used as the test samples. With these samples, a laboratory performance study of the DNA extraction and real-time PCR operations was conducted. This confirmed that the 18 participating laboratories were generally performing the DNA extraction and real-time PCR operations correctly. However, some laboratories using certain DNA amplification reagent with some real-time PCR instruments were not able to determine the PRSV-YK detection test. This suggests that the PRSV-YK detection test may not be able to correctly detect samples containing GM papaya when performed with these combinations of instruments and reagent. In order to ensure the reliability of the PRSV-YK detection test, it is necessary to examine in detail how the combination of DNA polymerase reagents and real-time PCR instruments affects the detection limit, and to implement an appropriate solution.

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan ® probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a crop fruit native to the USA and Mexico currently cultivated in several countries, including Brazil, Uruguay, Argentina, Chile, Peru, China, South Africa, and Australia. Supported by the increasing consumption and market prices, the interest in the cultivation of this fruit crop is strongly growing around the world. In this study, AFLP and S-SAP markers were employed to characterize the genetic diversity of ancient accessions of pecan from southern Brazil. The evaluated plants were selected and preserved by the farmers and are remnants of the first introduction of seedlings from the U.S.A into southern Brazil aiming at developing research towards establishing commercial orchards. High levels of genetic diversity were estimated, suggesting that these plants have an important genetic background for the establishment of a germplasm collection with a wide genetic basis, for the development of breeding programs for this fruit crop. Cluster analysis of the genetic datasets revealed some correlation between the nuts' morphometric traits and genetic markers. Such correlation should be further exploited. These ancient genotypes must be evaluated for other agronomic traits of interest and included in core collections of pecans.

Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Golden Camellias have recently been used as a food, cosmetic, and traditional medicine in China and Vietnam. Forty-two species have natural distribution in Vietnam, of which thirty-two species were considered endemic species of this country. The morphology of leaves and flowers of these species were similar; therefore, their taxonomic identification usually needed experts and the authentication has often been confused among species. Our study aims to describe the genetic diversity and the relationship of six species Camellia phanii, Camellia tamdaoensis, Camellia tienii, Camellia flava, Camellia petelotii and Camellia euphlebia by using three chloroplast DNA-barcodes: matK, rbcL and trnH-psbA. We also clarified the significant differences in anatomical characteristics of midvein and blade of their leaves, which suggested the possibility to use these criteria in taxonomy. In addition, preliminary chemical profiles of the methanolic extracts of leaves from six Golden Camellias such as total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC) and chlorogenic acids content (TCGAs) also showed the diversity among them. Interestingly, the discrimination on the catechins profile among six species followed the same tendency with the genetic distance on the phylogeny tree suggesting that catechins (i. e., discriminative catechins) can be biomarkers for the chemotaxonomy of these six Golden Camellias., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.

Background: Pachygenium embraces a group of terrestrial species formerly placed in Pelexia sensu lato. The genus currently comprises some 60 species, most of which are known from the southern parts of Brazil and Paraguay, with few species distributed in the Andean countries-only four species have been recorded from Argentina so far. In Jujuy Province, Argentina a new species of Pachygenium was found during our fieldwork. The aim of this article was to provide morphological and molecular evidence for its membership in this genus., Methods: Materials from specimens were collected in the field and examined by classical taxonomic and molecular biological techniques, e.g. , PCR and sequencing DNA. Phylogenetic reconstruction was performed by maximum-likelihood and Bayesian inference., Results: Pachygenium laurense from Argentina is described and illustrated based on morphological evidence and its taxonomic position was confirmed by phylogenetic analyses. A new combination for Pachygenium gutturosa is also proposed. A key for identification is provided for the Pachygenium species occurring in Argentina., Conclusion: Pachygenium laurense is the fifth species of the genus recorded from Argentina., Competing Interests: The authors declare there are no competing interests., (©2022 M. Martin et al.)

Pelargonium is a versatile genus mainly from the Cape Region, South Africa. The genus is divided into four subgenera and 16 sections characterized by several groups of chromosomes sizes and numbers. The DNA content of species from all subgenera and sections of Pelargonium, except for the sections Subsucculentia and Campylia was estimated using flow cytometry. Nuclei of Pelargonium samples (leaf or petal tissue) and an internal plant standard (leaf tissue) were isolated together and stained with propidium iodide. The DNA content was estimated providing that the 2C peaks of sample and standard be in linearity in the flow cytometer histograms. In total, 96 Pelargonium accessions of 60 species (22 Pelargonium species for the first time) were analyzed. The 2C DNA content ranged from 0.84 pg (P. longifolium, section Hoarea) to 6.69 pg (P. schizopetalum, section Magnistipulacea) and the corresponding 1Cx DNA content from 0.42 pg (P. longifolium) to 1.72 pg (P. transvaalense. This demonstrates the high plasticity within the genus Pelargonium. Some species, such as P. peltatum accessions revealed a pronounced endopolyploidization in leaves but not in petals underlining the importance to choose the right tissue as sample for the flow cytometry analysis. The reported genome sizes are a step forward towards the characterization of the Pelargonium collection within the German Gene Bank for Ornamental Plants and a valuable base for future sequencing programs of the Pelargonium genomes., Competing Interests: The authors have declared that no competing interests exist.

DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2 n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2 n chromosome numbers. The 2 n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora , while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea , E. catorce , E. juarezensis and E. perezcalixii . In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

Lavender species are widely distributed in their wild forms around the Mediterranean Basin and they are also cultivated worldwide as improved and registered clonal varieties. The economic interest of the species belonging to the Lavandula genus is determined by their use as ornamental plants and important source of essential oils that are destinated to the production of cosmetics, pharmaceuticals and foodstuffs. Because of the increasing number of cases of illegal commercialization of selected varieties, the protection of plant breeders' rights has become of main relevance for the recognition of breeding companies' royalties. With this aim, genomic tools based on molecular markers have been demonstrated to be very reliable and transferable among laboratories, and also much more informative than morphological descriptors. With the rising of the next-generation sequencing (NGS) technologies, several genotyping-by-sequencing approaches are now available. This study deals with a deep characterization of 15 varietal clones, belonging to two distinct Lavandula species, by means of restriction-site associated DNA sequencing (RAD-Seq). We demonstrated that this technology screens single nucleotide variants that enable to assess the genetic identity of individual accessions, to reconstruct genetic relationships among related breeding lines, to group them into genetically distinguishable main subclusters, and to assign their molecular lineages to distinct ancestors. Moreover, a number of polymorphic sites were identified within genes putatively involved in biosynthetic pathways related to both tissue pigmentation and terpene production, useful for breeding and/or protecting newly registered varieties. Overall, the results highlighted the presence of pure ancestries and interspecific hybrids for the analyzed Lavandula species, and demonstrated that RAD-Seq analysis is very informative and highly reliable for characterizing Lavandula clones and managing plant variety protection.

Bird nests in natural history collections are an abundant yet vastly underutilized source of genetic information. We sequenced the nuclear ribosomal internal transcribed spacer to identify plant species used as nest material in two contemporary (2003 and 2018) and two historical (both 1915) nest specimens constructed by Song Sparrows (Melospiza melodia) and Savannah Sparrows (Passerculus sandwichensis). A total of 13 (22%) samples yielded single, strong bands that could be identified using GenBank resources: six plants (Angiospermae), six green algae (Chlorophyta), and one ciliate (Ciliophora). Two native plant species identified in the nests included Festuca microstachys, which was introduced to the nest collection site by restoration practitioners, and Rosa californica, identified in a nest collected from a lost habitat that existed about 100 years ago. Successful sequencing was correlated with higher sample mass and DNA quality, suggesting future studies should select larger pieces of contiguous material from nests and materials that appear to have been fresh when incorporated into the nest. This molecular approach was used to distinguish plant species that were not visually identifiable, and did not require disassembling the nest specimens as is a traditional practice with nest material studies. The many thousands of nest specimens in natural history collections hold great promise as sources of genetic information to address myriad ecological questions., Competing Interests: The authors have declared that no competing interests exist.

In this study, we aim to develop a novel loop mediated isothermal amplification (LAMP) coupled with TaqMan (LAMP-TaqMan) method for quick qualitative detection of genetically modified organism (GMOs). We designed four LAMP primers and one TaqMan probe for the LAMP-TaqMan detection method to detect the nopaline synthase gene (NOS) terminator in GMOs. This assay enabled the amplification of DNA within ~20 min at a constant temperature of 65 °C. This assay detected as few as five copies of target sequences, which had a high specificity similar to the TaqMan qPCR method. Furthermore, the LAMP-TaqMan detection method was successfully used to amplify and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel LAMP-TaqMan assay has been developed, which has the similar sensitivity but takes less time than the TaqMan qPCR method. This method offers a novel approach for rapid detection of GMOs in foods., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Information obtained from the analysis of dust, particularly biological particles such as pollen, plant parts, and fungal spores, has great utility in forensic geolocation. As an alternative to manual microscopic analysis of dust components, we developed a pipeline that utilizes the airborne plant environmental DNA (eDNA) in settled dust to estimate geographic origin. Metabarcoding of settled airborne eDNA was used to identify plant species whose geographic distributions were then derived from occurrence records in the USGS Biodiversity in Service of Our Nation (BISON) database. The distributions for all plant species identified in a sample were used to generate a probabilistic estimate of the sample source. With settled dust collected at four U.S. sites over a 15-month period, we demonstrated positive regional geolocation (within 600 km 2 of the collection point) with 47.6% (20 of 42) of the samples analyzed. Attribution accuracy and resolution was dependent on the number of plant species identified in a dust sample, which was greatly affected by the season of collection. In dust samples that yielded a minimum of 20 identified plant species, positive regional attribution was achieved with 66.7% (16 of 24 samples). For broader demonstration, citizen-collected dust samples collected from 31 diverse U.S. sites were analyzed, and trace plant eDNA provided relevant regional attribution information on provenance in 32.2% of samples. This showed that analysis of airborne plant eDNA in settled dust can provide an accurate estimate regional provenance within the U.S., and relevant forensic information, for a substantial fraction of samples analyzed., (© 2021. The Author(s).)

Australia has over 30 Panicum spp. (panic grass) including several non-native species that cause crop and pasture loss and hepatogenous photosensitisation in livestock. It is critical to correctly identify them at the species level to facilitate the development of appropriate management strategies for efficacious control of Panicum grasses in crops, fallows and pastures. Currently, identification of Panicum spp. relies on morphological examination of the reproductive structures, but this approach is only useful for flowering specimens and requires significant taxonomic expertise. To overcome this limitation, we used multi-locus DNA barcoding for the identification of ten selected Panicum spp. found in Australia. With the exception of P. buncei, other native Australian Panicum were genetically separated at the species level and distinguished from non-native species. One nuclear (ITS) and two chloroplast regions (matK and trnL intron-trnF) were identified with varying facility for DNA barcode separation of the Panicum species. Concatenation of sequences from ITS, matK and trnL intron-trnF regions provided clear separation of eight regionally collected species, with a maximum intraspecific distance of 0.22% and minimum interspecific distance of 0.33%. Two of three non-native Panicum species exhibited a smaller genome size compared to native species evaluated, and we speculate that this may be associated with biological advantages impacting invasion of non-native Panicum species in novel locations. We conclude that multi-locus DNA barcoding, in combination with traditional taxonomic identification, provides an accurate and cost-effective adjunctive tool for further distinguishing Panicum spp. at the species level., (© 2021. The Author(s).)

Despite many attempts in the Sanger sequencing era, the phylogeny of fig trees remains unresolved, which limits our ability to analyze the evolution of key traits that may have contributed to their evolutionary and ecological success. We used restriction-site-associated DNA sequencing (c. 420 kb) and 102 morphological characters to elucidate the relationships between 70 species of Ficus. To increase phylogenetic information for higher-level relationships, we targeted conserved regions and assembled paired reads into long loci to enable the retrieval of homologous loci in outgroup genomes. We compared morphological and molecular results to highlight discrepancies and reveal possible inference bias. For the first time, we recovered a monophyletic subgenus Urostigma (stranglers) and a clade with all gynodioecious Ficus. However, we show, with a new approach based on iterative principal component analysis, that it is not (and will probably never be) possible to homogenize evolutionary rates and GC content for all taxa before phylogenetic inference. Four competing positions for the root of the molecular tree are possible. The placement of section Pharmacosycea as sister to other fig trees is not supported by morphological data and considered a result of a long-branch attraction artefact to the outgroups. Regarding morphological features and indirect evidence from the pollinator tree of life, the topology that divides Ficus into monoecious versus gynodioecious species appears most plausible. It seems most likely that the ancestor of fig trees was a freestanding tree and active pollination is inferred as the ancestral state, contrary to previous hypotheses. However, ambiguity remains on the ancestral breeding system. Despite morphological plasticity, we advocate restoring a central role to morphology in our understanding of the evolution of Ficus, as it can help detect systematic errors that appear more pronounced with larger molecular datasets., (© 2020 Willi Hennig Society.)

Tikal, a major city of the ancient Maya world, has been the focus of archaeological research for over a century, yet the interactions between the Maya and the surrounding Neotropical forests remain largely enigmatic. This study aimed to help fill that void by using a powerful new technology, environmental DNA analysis, that enabled us to characterize the site core vegetation growing in association with the artificial reservoirs that provided the city water supply. Because the area has no permanent water sources, such as lakes or rivers, these reservoirs were key to the survival of the city, especially during the population expansion of the Classic period (250-850 CE). In the absence of specific evidence, the nature of the vegetation surrounding the reservoirs has been the subject of scientific hypotheses and artistic renderings for decades. To address these hypotheses we captured homologous sequences of vascular plant DNA extracted from reservoir sediments by using a targeted enrichment approach involving 120-bp genetic probes. Our samples encompassed the time before, during and after the occupation of Tikal (1000 BCE-900 CE). Results indicate that the banks of the ancient reservoirs were primarily fringed with native tropical forest vegetation rather than domesticated species during the Maya occupation.

Cynara cardunculus L. is a cardoon species native to the Mediterranean region, which is composed of three botanical taxa, each having distinct biological characteristics. The aim of this study was to examine wild populations of C. cardunculus established in Portugal, in order to determine their genetic diversity, geographic distribution, and population structure. Based on SSR markers, 121 individuals of C. cardunculus from 17 wild populations of the Portuguese Alentejo region were identified and analysed. Ten SSRs were found to be efficient markers in the genetic diversity analysis. The total number of alleles ranged from 9 to 17 per locus. The expected and observed means in heterozygosity, by population analysed, were 0.591 and 0.577, respectively. The wild population exhibited a high level of genetic diversity at the species level. The highest proportion of genetic variation was identified within a geographic group, while variation was lower among groups. Geographic areas having highest genetic diversity were identified in Alvito, Herdade da Abóboda, Herdade da Revilheira and Herdade de São Romão populations. Moreover, significant genetic differentiation existed between wild populations from North-Alentejo geographic locations (Arraiolos, Évora, Monte da Chaminé) and Centro Hortofrutícola, compared with other populations. This study reports genetic diversity among a representative number of wild populations and genotypes of C. cardunculus from Portugal. These results will provide valuable information towards future management of C. cardunculus germplasm., Competing Interests: The authors have declared that no competing interests exist.

Studies along elevational gradients worldwide usually find the highest plant taxa richness in mid-elevation forest belts. Hence, an increase in upper elevation diversity is expected in the course of warming-related treeline rise. Here, we use a time-series approach to infer past taxa richness from sedimentary ancient DNA from the south-eastern Tibetan Plateau over the last ~18,000 years. We find the highest total plant taxa richness during the cool phase after glacier retreat when the area contained extensive and diverse alpine habitats (14-10 ka); followed by a decline when forests expanded during the warm early- to mid-Holocene (10-3.6 ka). Livestock grazing since 3.6 ka promoted plant taxa richness only weakly. Based on these inferred dependencies, our simulation yields a substantive decrease in plant taxa richness in response to warming-related alpine habitat loss over the next centuries. Accordingly, efforts of Tibetan biodiversity conservation should include conclusions from palaeoecological evidence.

Seven date palm seeds ( Phoenix dactylifera L.), radiocarbon dated from the fourth century BCE to the second century CE, were recovered from archaeological sites in the Southern Levant and germinated to yield viable plants. We conducted whole-genome sequencing of these germinated ancient samples and used single-nucleotide polymorphism data to examine the genetics of these previously extinct Judean date palms. We find that the oldest seeds from the fourth to first century BCE are related to modern West Asian date varieties, but later material from the second century BCE to second century CE showed increasing genetic affinities to present-day North African date palms. Population genomic analysis reveals that by ∼2,400 to 2,000 y ago, the P. dactylifera gene pool in the Eastern Mediterranean already contained introgressed segments from the Cretan palm Phoenix theophrasti , a crucial genetic feature of the modern North African date palm populations. The P. theophrasti introgression fraction content is generally higher in the later samples, while introgression tracts are longer in these ancient germinated date palms compared to modern North African varieties. These results provide insights into crop evolution arising from an analysis of plants originating from ancient germinated seeds and demonstrate what can be accomplished with the application of a resurrection genomics approach., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Summer warming is driving a greening trend across the Arctic, with the potential for large-scale amplification of climate change due to vegetation-related feedbacks [Pearson et al., Nat. Clim. Chang. (3), 673-677 (2013)]. Because observational records are sparse and temporally limited, past episodes of Arctic warming can help elucidate the magnitude of vegetation response to temperature change. The Last Interglacial ([LIG], 129,000 to 116,000 y ago) was the most recent episode of Arctic warming on par with predicted 21st century temperature change [Otto-Bliesner et al., Philos. Trans. A Math. Phys. Eng. Sci. (371), 20130097 (2013) and Post et al., Sci Adv (5), eaaw9883 (2019)]. However, high-latitude terrestrial records from this period are rare, so LIG vegetation distributions are incompletely known. Pollen-based vegetation reconstructions can be biased by long-distance pollen transport, further obscuring the paleoenvironmental record. Here, we present a LIG vegetation record based on ancient DNA in lake sediment and compare it with fossil pollen. Comprehensive plant community reconstructions through the last and current interglacial (the Holocene) on Baffin Island, Arctic Canada, reveal coherent climate-driven community shifts across both interglacials. Peak LIG warmth featured a ∼400-km northward range shift of dwarf birch, a key woody shrub that is again expanding northward. Greening of the High Arctic-documented here by multiple proxies-likely represented a strong positive feedback on high-latitude LIG warming. Authenticated ancient DNA from this lake sediment also extends the useful preservation window for the technique and highlights the utility of combining traditional and molecular approaches for gleaning paleoenvironmental insights to better anticipate a warmer future., Competing Interests: The authors declare no competing interest.

Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals' protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F 3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F 3:4 and F 3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.

The free online trading of herbal mixtures useful for various purposes facilitates the circulation of dangerous herbs or plant parts. This is the case, for example, of the illegal trade in seeds of Peganum harmala ( Pgh ), which contain alkaloids capable of inhibiting monoamine oxidase (MAO) and are therefore used in hallucinogenic preparations, such as the psychedelic drink ayahuasca . The precise identification of these seeds and their distinction from other very similar but not dangerous seeds are necessary for forensic purposes and represents an advance in avoiding the adulteration of mixtures. In this work, we show the qualitative identification of Pgh seeds by optical and electron microscopy and the parallel development of a real-time qPCR test, which reveals, in a species-specific manner, the presence of Pgh DNA up to quantities lower than 1 pg. In addition to the species specificity and high sensitivity, the reaction accurately quantifies the presence of seeds or parts of seeds of Pgh in complex herbal mixtures, thus giving an indication of the danger or otherwise of the product.

Regarding the prospective investigation of food authenticity and adulteration the aim of the present study was the development and validation of a real-time PCR assay to identify hemp (Cannabis sativa) which has gained increasing importance as a valuable food ingredient. The assay targets a specific spacer DNA sequence in Cannabis sativa chloroplasts and detects 1.5 pg hemp DNA, which is equivalent to 18 copies/µL. Corresponding to the very low LOD (0.00031 ng/µL) the method allows the detection of hemp even in the infinitesimal concentration of contaminants. Due to a SNP in position 603, hemp can be identified unequivocally and discriminated from its closest relative hops (Humulus lupulus). The PCR method shows no cross-reactivity with 39 of 46 tested plant species. Low cross-reactivity with mulberry, stinging nettle, lavender, cornflower, wine, figs and hops can be neglected, because the Δ Ct-values are > 14, and the obtained Ct-values are beyond the cut-off for a positive assessment (Ct-values ≤ 33). Moreover, the suitability of the method to identify hemp as a food ingredient was proved by analysing diverse food products such as chocolate or cookies.

Here, we describe DNA enrichment of the zein gene from maize using pyrrolidinyl peptide nucleic acid (PNA) immobilized on a magnetic solid support as a capture element. Magnetite nanoparticles (MNP) with a capacity of 373 pmolPNA/mg and coated with poly(N-acryloylglycine) (PNAG) showed a good response to magnetic field. The PNA probe immobilized on the MNP discriminated between non-complementary and complementary DNA using fluorophore-tagged DNA as a model. We applied this system for the enrichment of the zein gene from maize in eight cereal product samples. After DNA desorption from the MNP, and its amplification via polymerase chain reaction (PCR), gel electrophoresis indicated that only cereal samples containing the zein gene from maize yielded positive results, indicating a high binding specificity between the PNA used and the complementary DNA. This PNA-functionalized MNP is potentially useful as an effective nano-solid support for DNA enrichment from other samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Interactions between plants and insects are among the most important life functions for all organism at a particular natural community. Usually a large number of samples are required to identify insect diets in food web studies. Previously, Sanger sequencing and next generation sequencing (NGS) with short DNA barcodes were used, resulting in low species-level identification; meanwhile the costs of Sanger sequencing are expensive for metabarcoding together with more samples. Here, we present a fast and effective sequencing strategy to identify larvae of Lepidoptera and their diets at the same time without increasing the cost on Illumina platform in a single HiSeq run, with long-multiplex-metabarcoding (COI for insects, rbcL, matK, ITS and trnL for plants) obtained by Trinity assembly (SHMMT). Meanwhile, Sanger sequencing (for single individuals) and NGS (for polyphagous) were used to verify the reliability of the SHMMT approach. Furthermore, we show that SHMMT approach is fast and reliable, with most high-quality sequences of five DNA barcodes of 63 larvae individuals (54 species) recovered (full length of 100% of the COI gene and 98.3% of plant DNA barcodes) using Trinity assembly (up-sized to 1015 bp). For larvae diets identification, 95% are reliable; the other 5% failed because their guts were empty. The diets identified by SHMMT approach are 100% consistent with the host plants that the larvae were feeding on during our collection. Our study demonstrates that SHMMT approach is reliable and cost-effective for insect-plants network studies. This will facilitate insect-host plant studies that generally contain a huge number of samples., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

The global climate is changing, resulting in significant economic losses worldwide. It is thus necessary to speed up the plant selection process, especially for complex traits such as biotic and abiotic stresses. Nowadays, genomic selection (GS) is paving new ways to boost plant breeding, facilitating the rapid selection of superior genotypes based on the genomic estimated breeding value (GEBV). GEBVs consider all markers positioned throughout the genome, including those with minor effects. Indeed, although the effect of each marker may be very small, a large number of genome-wide markers retrieved by high-throughput genotyping (HTG) systems (mainly genotyping-by-sequencing, GBS) have the potential to explain all the genetic variance for a particular trait under selection. Although several workflows for GBS and GS data have been described, it is still hard for researchers without a bioinformatics background to carry out these analyses. This chapter has outlined some of the recently available bioinformatics resources that enable researchers to establish GBS applications for GS analysis in laboratories. Moreover, we provide useful scripts that could be used for this purpose and a description of key factors that need to be considered in these approaches.

Transposable elements (TEs) are ubiquitous repetitive components of eukaryotic organisms that show mobility in the genome against diverse stresses. TEs contribute considerably to the size, structure, and plasticity of genomes and also play an active role in genome evolution by helping their hosts adapt to novel conditions by conferring useful characteristics. We developed a simple and rapid method for investigation of genetic mobility and diversity among TEs in combination with a target region amplification polymorphism (TE-TRAP) marker system in gamma-irradiated sorghum mutants. The TE-TRAP marker system reveals a high level of genetic diversity, which provides a useful marker resource for genetic mobility research.

Forward genetic analysis remains as one of the most powerful tools for assessing gene functions, although the identification of the causal mutation responsible for a given phenotype has been a tedious and time-consuming task until recently. Advances in deep sequencing technologies have provided new approaches for the exploitation of natural and artificially induced genetic diversity, thus accelerating the discovery of novel allelic variants. In this chapter, a mapping-by-sequencing forward genetics approach is described to identify causal mutations in tomato (Solanum lycopersicum L.), a major crop species that is also a model species for plant biology and breeding.

High-resolution melting (HRM) analysis is a cost-effective, specific, and rapid tool that allows distinguishing genetically related plants and other organisms based on the detection of small nucleotide variations, which are recognized from melting properties of the double-stranded DNA. It has been widely applied in several areas of research and diagnostics, including botanical authentication of several food commodities and herbal products. Generally, it consists of the main steps: (1) in silico sequence analysis and primer design; (2) DNA extraction from plant material; (3) amplification by real-time PCR with an enhanced fluorescent dye targeting a specific DNA barcode or other regions of taxonomic interest (100-200 bp); (4) melting curve analysis; and (5) statistical data analysis using a specific HRM software. This chapter presents an overview of HRM analysis and application, followed by the detailed description of all the required reagents, instruments, and protocols for the successful and easy implementation of a HRM method to differentiate closely related plant species.

Quantitative trait loci mapping has become a common practice in crop plants and can be accomplished using either biparental populations following interval mapping or natural populations following the approach of association mapping. Because of its ability to use the natural diversity and to search for functional variants in a broader germplasm, association mapping is becoming popular among researchers. An overview of the different steps involved in association mapping in plants is provided in this chapter.

Transposable elements (TEs) are mobile, recurring DNA sequences scattered throughout genome and have a large impact on genome structure and function. Several genetic marker techniques were developed to exploit their ubiquitous nature. Sequence-specific amplified polymorphism (SSAP) is a TE-based genetic marker system that has been used in various purposes such as measuring genetic relatedness between species, deciphering the population structures, molecular tagging for agronomic development in marker-assisted breeding (MAS). In addition to SSAP, sequence characterized amplified region (SCAR) from the SSAP markers provides an added advantage in identifying qualitative traits. Once developed SCAR markers are efficient, fast, and reliable method for genetic evaluations. These methods can be useful especially for the crops which have no genetic sequence information. With improved discriminatory ability they offer access to dynamic and polymorphic regions of genome. These techniques can be useful in breeding programs to improve or develop high yielding crops.

Isolating high-quality DNA is essential for several applications in molecular biology and genomics. Performing whole-genome sequencing in crops and development of reduced representation genomic libraries for genotyping require precise standard on DNA in terms of concentration and purity. For screening large populations it is essential to increase the extraction throughput at affordable costs. In this chapter a homemade protocol is provided that is able to isolate in 96-well plates 198 samples of DNA in a single extraction. The method has been validated in tomato and pepper and can be applied in several vegetable species.

Most plant agronomic traits are quantitatively inherited. Identification of quantitative trait loci (QTL) is a challenging target for most scientists and crop breeders as large-scale genotyping is difficult. Molecular marker technology has continuously evolved from hybridization-based technology to PCR-based technology, and finally, sequencing-based high-throughput single-nucleotide polymorphisms (SNPs). High-throughput sequencing technologies can provide strategies for sequence-based SNP genotyping. Here we describe the SLAF-seq that can be applied as the SNP genotyping approach. The high-throughput SNP genotyping methods will prove useful for the construction of high-density genetic maps and identification of QTLs for their deployment in plant breeding and facilitate genome-wide selection (GWS) and genome-wide association studies (GWAS).

<b>Background and Objective:</b> Protocols commonly used in plant DNA extraction were known to be highly time-consuming and harmful due to the application of some hazardous reagents. Therefore, it was not applicable for such laboratories with limited resources as well as for high-throughput analysis. This study was aimed to develop a rapid yet less hazardous DNA extraction protocol for a plant using potassium phosphate buffer. <b>Materials and Methods:</b> Genomic DNA of chili pepper (<i>Capsicum annuum</i>) was extracted using potassium phosphate buffer and its efficacy was compared to three widely known protocols (CTAB-based, mini preparation and commercial kit). The extracted DNA from those four methods was evaluated based on its quality, quantity, practicality and cost per reaction. <b>Results:</b> Genomic DNA resulted from potassium phosphate buffer-based protocol exhibited comparable quality with adequate concentration for further downstream analysis. Results of PCR and sequencing were also emphasized the amplifiable DNA quality from this developed protocol. Compared to those commonly used protocols, potassium phosphate buffer consisted of 5 main working steps only, thus providing a simple yet rapid plant DNA extraction protocol. Since this protocol used ethanol only, it also offered a less hazardous and low-cost protocol that applicable for those resource-limited laboratories. <b>Conclusion:</b> This developed protocol provided a promising alternative of plant DNA extraction that might be applicable for both large scale analysis and any laboratory type. Further investigation was needed to evaluate its efficacy in extracting genomic DNA from various plants with different morphological characteristic.

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

One-pot synthesis of novel hydrogel-based anion exchange membranes (AEMs), with only a single-phase monomer mixture, was used to eliminate surface heterogeneity and generate reproducible electroconvective microvortices in the over-limiting region of the current-voltage characteristic (CVC) curves. Diallyldimethylammonium chloride (DDA) was used as the main component to provide the cation charge groups, and 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethyl acrylate (EGDMA) were used as the auxiliary structure monomers. The uniform membrane structure allowed reproducible and sensitive DNA detection and quantification, as probe-target surface complexes can gate the ion flux and produce large voltage shifts in the over-limiting region. Suppressed membrane curvature due to controlled swelling is a crucial part to avoid the reduction of depletion region for maintaining the influence of target gene hybridization. Fourier-transform infrared (FTIR) spectroscopy verified the synthesized membrane structure, with a residual vinyl group that allows easy carboxylation via additional photografting reaction. Consequently, a significantly higher DNA probe functionalization efficiency is obtained on the homogeneous AEMs, evidenced by the increasing nitrogen element content and bonding via X-ray photoelectron spectroscopy (XPS). The DDA content was optimized to provide a sufficient coulomb force between AEM and nucleic acid backbone to promote the specific binding efficiency but without high dimensional swelling which might change the surface geometry and restrict the voltage shifting for sensing in the over-limiting region, and the optimal DDA/HEMA ratio was found to be 4/10. The synthesized AEM sensor for recombinant 35S promoter sequence identification exhibited a reproducible calibration standard curve with dynamic range between 30 fM and 1 μM and high selectivity with only 0.01 V shift for 1 μM nontarget oligo.

White clover (Trifolium repens L.) is an important perennial legume forage with high productivity and quality. To strengthen the basic research on the genetic characteristics, fingerprint identification and adaptability of white clover germplasm resources, Simple sequence repeat (SSR) molecular markers were applied to 10 white clover cultivars to assess the genetic diversity and related lines of white clover at the molecular level in order to lay a theoretical foundation for the selection of high-quality seeds and cultivars of white clover. A total of 120 different bands were amplified by 29 pairs of SSR primers with good polymorphism, of which 103 (89.5%) were polymorphic. Meanwhile, the PIC of each primer was 0.181-0.588, with an average of 0.329. Analysis of molecular variance revealed that 57% of the genetic variation occurred within cultivars and 43% occurred among cultivars. The results of cluster analysis and the principal coordinate analysis revealed that the parental relationships of the 10 cultivars, with the 'Purple' cultivar very distantly related to the other 9 cultivars and the closest parental relationship between 'Ladino' and 'Sulky'. The fingerprints constructed by three representative primers (gtrs679, gtrs319, and gtrs678) have a strong identification ability. In summary, the SSR markers had good polymorphism and could be used for DNA fingerprint analysis of white clover cultivars.

Satellite DNA (satDNA) is one of the major fractions of the eukaryotic nuclear genome. Highly variable satDNA is involved in various genome functions, and a clear link between satellites and phenotypes exists in a wide range of organisms. However, little is known about the origin and temporal dynamics of satDNA. The "library hypothesis" indicates that the rapid evolutionary changes experienced by satDNAs are mostly quantitative. Although this hypothesis has received some confirmation, a number of its aspects are still controversial. A recently developed next-generation sequencing (NGS) method allows the determination of the satDNA landscape and could shed light on unresolved issues. Here, we explore low-coverage NGS data to infer satDNA evolution in the phylogenetic context of the diploid species of the Chenopodium album aggregate. The application of the Illumina read assembly algorithm in combination with Oxford Nanopore sequencing and fluorescent in situ hybridization allowed the estimation of eight satDNA families within the studied group, six of which were newly described. The obtained set of satDNA families of different origins can be divided into several categories, namely group-specific, lineage-specific and species-specific. In the process of evolution, satDNA families can be transmitted vertically and can be eliminated over time. Moreover, transposable element-derived satDNA families may appear repeatedly in the satellitome, creating an illusion of family conservation. Thus, the obtained data refute the "library hypothesis", rather than confirming it, and in our opinion, it is more appropriate to speak about "the library of the mechanisms of origin"., Competing Interests: The authors have declared that no competing interests exist.

Traditional medicines are widely traded across the globe and have received considerable attention in the recent past, with expectations of heightened demand in the future. However, there are increasing global concerns over admixture, which can affect the quality, safety, and efficacy of herbal medicinal products. In this study, we aimed to use DNA metabarcoding to identify 39 Thai herbal products on the Thai National List of Essential Medicines (NLEM) and assess species composition and admixture. Among the products, 24 samples were in-house-prepared formulations, and 15 samples were registered formulations. In our study, DNA metabarcoding analysis using ITS2 and rbcL barcode regions were employed to identify herbal ingredients mentioned in the products. The nuclear region, ITS2, was able to identify herbal ingredients in the products at the genus- and family-levels in 55% and 63% of cases, respectively. The chloroplast gene, rbcL, enabled genus- and family-level identifications in 58% and 73% of cases, respectively. In addition, plant species were detected in larger numbers (Family identified, absolute %) in registered herbal products than in in-house-prepared formulations. The level of fidelity increases concerns about the reliability of the products. This study highlights that DNA metabarcoding is a useful analytical tool when combined with advanced chemical techniques for the identification of plant species in highly processed, multi-ingredient herbal products.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used since ancient times as culinary herbs and remedies in traditional medicine. Although it is a relatively small genus, intra-generic species delineation, as well as its inter-generic relationships within tribe Mentheae, are still poorly understood. High resolution melting (HRM) analysis, coupled with microsatellite markers (SSRs), could facilitate the molecular identification and characterization of certain genotypes more efficiently and relatively faster when compared to other analytical methods. In this study, 38 Origanum samples corresponding to six Origanum taxa (O. dictamnus, O. majorana, O. onites, O. scabrum, O. sipyleum, and O. vulgare subsp. hirtum) were analyzed, using six microsatellite loci. Our goal was to molecularly identify and discriminate among the selected samples and to evaluate the ability of the HRM technique as an analytical tool for the discrimination of Origanum species from Greece. The temperature-shifted melting curves produced by the HRM analysis, resulted in 98 unique HRM profiles, which enabled the discrimination of the Origanum genotypes studied. According to the similarity dendrogram based on the HRM profiles, six unique clusters were formed, each one corresponding to a single taxon. In conclusion, HRM genotyping provided a fast, cost-effective method, well suited for the molecular characterization and identification of Origanum taxa and for the authentication of the original genetic material.