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.

Old but still good: genetic diversity of ancient pecan genotypes from southern Brazil.

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.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

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.

Development of a specific and sensitive diagnostic PCR for rapid molecular authentication of the medicinal plant Portulaca oleracea.

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.

Genotypic and Phenotypic Diversity of Endemic Golden Camellias Collected from the North of Vietnam.

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.

Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

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.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

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.

DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues.

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.

Pachygenium laurense (Orchidaceae, Spiranthinae), a new orchid species from Argentina-morphological evidence and phylogenetic reconstruction.

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.

The variability of nuclear DNA content of different Pelargonium species estimated by flow cytometry.

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.

Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae).

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 2n 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 2n chromosome numbers. The 2n 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.

Genotyping Analysis by RAD-Seq Reads Is Useful to Assess the Genetic Identity and Relationships of Breeding Lines in Lavender Species Aimed at Managing Plant Variety Protection.

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 as botanical time capsules: DNA barcoding identifies the contents of contemporary and historical nests.

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.

Exploring systematic biases, rooting methods and morphological evidence to unravel the evolutionary history of the genus Ficus (Moraceae).

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.

Genotypic identification of Panicum spp. in New South Wales, Australia using DNA barcoding.

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.

Geographic source estimation using airborne plant environmental DNA in dust.

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 km2 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.

Environmental DNA reveals arboreal cityscapes at the Ancient Maya Center of Tikal.

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.

Genetic diversity and population structure of Cynara cardunculus L. in southern Portugal.

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.

The genomes of ancient date palms germinated from 2,000 y old seeds.

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.