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1.
PLoS One ; 15(2): e0228624, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101546

RESUMO

We report a rapid and accurate quantitative detection method using droplet digital PCR (ddPCR) technology to identify cassava adulteration in starch products. The ddPCR analysis showed that the weight of cassava (M) and cassava-extracted DNA content had a significant linear relationship-the correlation coefficient was R2 = 0.995, and the maximum coefficient of variation of replicates was 7.48%. The DNA content and DNA copy number (C) measured by ddPCR also had a linear relationship with R2 = 0.992; the maximum coefficient of variation of replicates was 8.85%. The range of cassava ddPCR DNA content was 25 ng/µL, and the formula M = (C + 32.409)/350.579 was obtained by converting DNA content into the median signal. The accuracy and application potential of the method were verified using the constructed adulteration model.


Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos , Manihot/química , Reação em Cadeia da Polimerase/métodos , Amido/química , DNA de Plantas/análise , DNA de Plantas/genética , Análise de Alimentos/normas , Manihot/genética , Reação em Cadeia da Polimerase/normas , Amido/normas
2.
Anal Bioanal Chem ; 412(7): 1701-1707, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31965247

RESUMO

Two "mass ratio-DNA copy concentration ratio" formulas were established respectively on droplet digital PCR (ddPCR) and chip-based digital PCR (cdPCR) to determine the mass ratio of kidney bean, a common alternative plant-derived ingredient in lotus seed paste. The limit of detection for DNA copy concentration of kidney bean and lotus seed was 6 copies/µL. Quantitative detection range was set from 5 to 80%, and the limit of quantification for mass ratio of kidney bean in lotus seed paste was defined as 5%. Results of 6 simulated samples and 16 prepackaged pastes in this work offer compelling evidence that an innovative scheme for quantitative detection of kidney bean in lotus seed paste was available, and provide technical support for the identification of suspicious ingredients from fraudulent substitution or adventitious contamination. Graphical abstract Two "mass ratio-DNA copy concentration ratio" formulas were established respectively on droplet digital PCR (ddPCR) and chip digital PCR (cdPCR) to determine the mass ratio of kidney bean in adulterated lotus seed paste. It was the first time to quantify adulterate food by directly converting DNA copy concentration ratio obtained from digital PCR to mass ratio, which could provide strong technical support for quantitative detection of adulterated food.


Assuntos
Contaminação de Alimentos/análise , Lotus/embriologia , Phaseolus , Reação em Cadeia da Polimerase/métodos , Sementes , Variações do Número de Cópias de DNA , DNA de Plantas/análise , Genes de Plantas , Limite de Detecção , Lotus/genética , Phaseolus/genética , Reprodutibilidade dos Testes
3.
Sensors (Basel) ; 19(21)2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31689974

RESUMO

Volatile organic compounds (VOCs) released by plants are closely associated with plant metabolism and can serve as biomarkers for disease diagnosis. Huanglongbing (HLB), also known as citrus greening or yellow shoot disease, is a lethal threat to the multi-billion-dollar citrus industry. Early detection of HLB is vital for removal of susceptible citrus trees and containment of the disease. Gas sensors are applied to monitor the air quality or toxic gases owing to their low-cost fabrication, smooth operation, and possible miniaturization. Here, we report on the development, characterization, and application of electrical biosensor arrays based on single-walled carbon nanotubes (SWNTs) decorated with single-stranded DNA (ssDNA) for the detection of four VOCs-ethylhexanol, linalool, tetradecene, and phenylacetaldehyde-that serve as secondary biomarkers for detection of infected citrus trees during the asymptomatic stage. SWNTs were noncovalently functionalized with ssDNA using π-π interaction between the nucleotide and sidewall of SWNTs. The resulting ssDNA-SWNT hybrid structure and device properties were investigated using Raman spectroscopy, ultraviolet (UV) spectroscopy, and electrical measurements. To monitor changes in the four VOCs, gas biosensor arrays consisting of bare SWNTs before and after being decorated with different ssDNA were employed to determine the different concentrations of the four VOCs. The data was processed using principal component analysis (PCA) and neural net fitting (NNF).


Assuntos
Biomarcadores/análise , Técnicas Biossensoriais/instrumentação , Citrus/metabolismo , DNA de Cadeia Simples/química , Nanotubos de Carbono/química , Doenças das Plantas , Árvores/metabolismo , Compostos Orgânicos Voláteis/análise , DNA de Plantas/análise , Análise de Componente Principal , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta , Análise Espectral Raman
4.
Proc Natl Acad Sci U S A ; 116(47): 23588-23593, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685619

RESUMO

A major challenge in biology is to understand how phylogeny, diet, and environment shape the mammalian gut microbiome. Yet most studies of nonhuman microbiomes have relied on relatively coarse dietary categorizations and have focused either on individual wild populations or on captive animals that are sheltered from environmental pressures, which may obscure the effects of dietary and environmental variation on microbiome composition in diverse natural communities. We analyzed plant and bacterial DNA in fecal samples from an assemblage of 33 sympatric large-herbivore species (27 native, 6 domesticated) in a semiarid East African savanna, which enabled high-resolution assessment of seasonal variation in both diet and microbiome composition. Phylogenetic relatedness strongly predicted microbiome composition (r = 0.91) and was weakly but significantly correlated with diet composition (r = 0.20). Dietary diversity did not significantly predict microbiome diversity across species or within any species except kudu; however, diet composition was significantly correlated with microbiome composition both across and within most species. We found a spectrum of seasonal sensitivity at the diet-microbiome nexus: Seasonal changes in diet composition explained 25% of seasonal variation in microbiome composition across species. Species' positions on (and deviations from) this spectrum were not obviously driven by phylogeny, body size, digestive strategy, or diet composition; however, domesticated species tended to exhibit greater diet-microbiome turnover than wildlife. Our results reveal marked differences in the influence of environment on the degree of diet-microbiome covariation in free-ranging African megafauna, and this variation is not well explained by canonical predictors of nutritional ecology.


Assuntos
Animais Selvagens/microbiologia , Dieta , Microbioma Gastrointestinal , Mamíferos/microbiologia , Animais , Animais Domésticos/microbiologia , Animais Domésticos/fisiologia , Animais Selvagens/fisiologia , DNA Bacteriano/análise , DNA de Plantas/análise , Fezes/química , Fezes/microbiologia , Herbivoria , Quênia , Mamíferos/fisiologia , Modelos Biológicos , Filogenia , Plantas Comestíveis , Ruminantes/microbiologia , Ruminantes/fisiologia , Estações do Ano , Especificidade da Espécie
5.
PLoS One ; 14(9): e0222707, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31536553

RESUMO

To determine the origin and genetic consequences of anagenesis in Rubus takesimensis on Ulleung Island, Korea, we compared the genetic diversity and population structure of R. takesimensis with those of its continental progenitor R. crataegifolius. We broadly sampled a total of 315 accessions in 35 populations and sequenced five noncoding regions of chloroplast DNA. Rubus takesimensis emerged as nonmonophyletic and several geographically diverse continental populations were likely responsible for the origin of R. takesimensis; the majority of R. takesimensis accessions were sisters to the clade containing accessions of R. crataegifolius, primarily from the Korean peninsula, while rare accessions from three populations shared common ancestors with the ones from the southern part of the Korean peninsula, Jeju Island, and Japan. A few accessions from the Chusan population originated independently from the Korean peninsula. Of 129 haplotypes, 81 and 48 were found exclusively in R. crataegifolius and R. takesimensis, respectively. We found unusually high genetic diversity in two regions on Ulleung Island and no geographic population structure. For R. crataegifolius, two major haplotype groups were found; one for the northern mainland Korean peninsula, and the other for the southern Korean peninsula and the Japanese archipelago. Compared with populations of R. crataegifolius sampled from Japan, much higher haplotype diversity was found in populations from the Korean peninsula. The patterns of genetic consequences in R. takesimensis need to be verified for other endemic species based on chloroplast DNA and independent nuclear markers to synthesize emerging patterns of anagenetic speciation on Ulleung Island.


Assuntos
Especiação Genética , Variação Genética , Haplótipos , Rubus/genética , DNA de Cloroplastos/análise , DNA de Cloroplastos/genética , DNA de Plantas/análise , DNA de Plantas/genética , Genética Populacional , Geografia , Ilhas , Japão , Filogenia , República da Coreia , Rubus/classificação , Rubus/crescimento & desenvolvimento , Análise de Sequência de DNA , Especificidade da Espécie
6.
BMC Evol Biol ; 19(1): 170, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412772

RESUMO

BACKGROUND: In the absence of sex and recombination, genomes are expected to accumulate deleterious mutations via an irreversible process known as Muller's ratchet, especially in the case of polyploidy. In contrast, no genome-wide mutation accumulation was detected in a transcriptome of facultative apomictic, hexaploid plants of the Ranunculus auricomus complex. We hypothesize that mutations cannot accumulate in flowering plants with facultative sexuality because sexual and asexual development concurrently occurs within the same generation. We assume a strong effect of purging selection on reduced gametophytes in the sexual developmental pathway because previously masked recessive deleterious mutations would be exposed to selection. RESULTS: We test this hypothesis by modeling mutation elimination using apomictic hexaploid plants of the R. auricomus complex. To estimate mean recombination rates, the mean number of recombinants per generation was calculated by genotyping three F1 progeny arrays with six microsatellite markers and character incompatibility analyses. We estimated the strength of purging selection in gametophytes by calculating abortion rates of sexual versus apomictic development at the female gametophyte, seed and offspring stage. Accordingly, we applied three selection coefficients by considering effects of purging selection against mutations on (1) male and female gametophytes in the sexual pathway (additive, s = 1.000), (2) female gametophytes only (s = 0.520), and (3) on adult plants only (sporophytes, s = 0.212). We implemented recombination rates into a mathematical model considering the three different selection coefficients, and a genomic mutation rate calculated from genome size of our plants and plant-specific mutation rates. We revealed a mean of 6.05% recombinants per generation. This recombination rate eliminates mutations after 138, 204 or 246 generations, depending on the respective selection coefficients (s = 1.000, 0.520, and 0.212). CONCLUSIONS: Our results confirm that the empirically observed frequencies of facultative recombination suffice to prevent accumulation of deleterious mutations via Muller's ratchet even in a polyploid genome. The efficiency of selection is in flowering plants strongly increased by acting on the haplontic (reduced) gametophyte stage.


Assuntos
Acúmulo de Mutações , Ranunculus/genética , Recombinação Genética , DNA de Plantas/análise , DNA de Plantas/genética , Repetições de Microssatélites , Taxa de Mutação , Óvulo Vegetal , Poliploidia , Ranunculus/crescimento & desenvolvimento , Ranunculus/fisiologia , Reprodução Assexuada
7.
Anal Chim Acta ; 1078: 24-31, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358225

RESUMO

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe3O4@Au magnetic nanoparticles via an Au-S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase. Finally, [Ru(NH3)6]3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction. The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH3)6]3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol L-1, with a detection limit of 4.5 × 10-17 mol L-1. Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity, stability, and reproducibility.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Plantas/análise , Técnicas Eletroquímicas/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Sequência de Bases , Calibragem , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Nanopartículas de Magnetita/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos Testes , Compostos de Rutênio/química
8.
BMC Plant Biol ; 19(1): 230, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151385

RESUMO

BACKGROUND: Interspecific hybridisation resulting in polyploidy is one of the major driving forces in plant evolution. Here, we present data from the molecular cytogenetic analysis of three cytotypes of Elytrigia ×mucronata using sequential fluorescence (5S rDNA, 18S rDNA and pSc119.2 probes) and genomic in situ hybridisation (four genomic probes of diploid taxa, i.e., Aegilops, Dasypyrum, Hordeum and Pseudoroegneria). RESULTS: The concurrent presence of Hordeum (descended from E. repens) and Dasypyrum + Aegilops (descended from E. intermedia) chromosome sets in all cytotypes of E. ×mucronata confirmed the assumed hybrid origin of the analysed plants. The following different genomic constitutions were observed for E. ×mucronata. Hexaploid plants exhibited three chromosome sets from Pseudoroegneria and one chromosome set each from Aegilops, Hordeum and Dasypyrum. Heptaploid plants harboured the six chromosome sets of the hexaploid plants and an additional Pseudoroegneria chromosome set. Nonaploid cytotypes differed in their genomic constitutions, reflecting different origins through the fusion of reduced and unreduced gametes. The hybridisation patterns of repetitive sequences (5S rDNA, 18S rDNA, and pSc119.2) in E. ×mucronata varied between and within cytotypes. Chromosome alterations that were not identified in the parental species were found in both heptaploid and some nonaploid plants. CONCLUSIONS: The results confirmed that both homoploid hybridisation and heteroploid hybridisation that lead to the coexistence of four different haplomes within single hybrid genomes occur in Elytrigia allopolyploids. The chromosomal alterations observed in both heptaploid and some nonaploid plants indicated that genome restructuring occurs during and/or after the hybrids arose. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile. Hybridisation in Triticeae allopolyploids clearly and significantly contributes to genomic diversity. Different combinations of parental haplomes coupled with chromosomal alterations may result in the establishment of unique lineages, thus providing raw material for selection.


Assuntos
Genótipo , Hibridização Genética , Poaceae/genética , Poliploidia , Análise Citogenética , República Tcheca , DNA de Plantas/análise , Hibridização In Situ , Hibridização in Situ Fluorescente , RNA Ribossômico 18S/análise , RNA Ribossômico 5S/análise
9.
Biosens Bioelectron ; 141: 111409, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31207569

RESUMO

The steady increase in commercialization of genetically modified organisms (GMOs) demands low-cost, rapid and portable GMO-detection methods that are technically and economically sustainable. Traditional nucleic acid detection platforms are still expensive, immobile and generate complex read-outs to be analyzed by experienced personal. Herein, we report the development of a portable, rapid and user-friendly GMO-detection biosensor, DaimonDNA. The system specifically amplifies the target DNA using loop-mediated isothermal amplification (LAMP) and provides real-time, naked-eye detection with Hydroxynaphthol blue reagent in less than 30 min. The construction of the platform relies on 3D printing and off-the-shelf electronic components that makes it extremely low-cost (<25 Euro), light weight (108 g), mobile (6 × 6 × 3 cm) and suitable for field deployment. We present the detection of the soybean lectin gene as a species control, and P35S as a transgene element found in many GMO varieties. We confirmed specificity of the DaimonDNA biosensor using" RoundUp Ready (RRS)" and MON89788 soybean genomic DNA with P35S and lectin primer sets. We characterized sensitivity of our system using 76.92, 769.2 and 7692 copies of RRS soybean genomic DNA in a non-GMO background. We benchmarked the DNA amplification and detection efficiency of our system against a thermocycling machine by quantifying the images obtained from gel electrophoresis and showed that our system is comparable to most other reported isothermal amplification techniques. This system can also be used for widespread point-of-care or field-based testing that is infrequently performed due to the lack of rapid, inexpensive, user-friendly and portable methods.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Soja/genética , Colorimetria/instrumentação , Primers do DNA/química , Primers do DNA/genética , DNA de Plantas/análise , Desenho de Equipamento , Naftalenossulfonatos/análise , Impressão Tridimensional , Transgenes
10.
J AOAC Int ; 102(6): 1798-1807, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31113529

RESUMO

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.


Assuntos
Camellia sinensis/química , Dano ao DNA , DNA de Plantas/análise , DNA de Plantas/genética , Extratos Vegetais/análise , Folhas de Planta/química , Manipulação de Alimentos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
11.
Methods Mol Biol ; 1991: 79-90, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041765

RESUMO

Plants, when challenged with any unfavorable condition, such as biotic or abiotic stress, adapt to the stress via physiological or structural changes. DNA methylation, an important epigenetic factor, plays an integral role in determining chromatin dynamicity and in turn regulates the process of gene transcription in eukaryotes. DNA methylation resulting in 5-methylcytosine interferes with the transcription process by hindering accessibility of the transcriptional machinery. Transcriptionally active genes are predominantly hypomethylated, whereas repressed genes exhibit hypermethylation. It can thus be interpreted that the presence of methylation in the promoter and upstream regions of loci represses their transcription and vice versa. Chop-PCR is a targeted DNA methylation detection technique that uses partial digestion by methylation-sensitive restriction enzymes (MSREs) followed by PCR amplification. The presence of cytosine methylation at the cleavage sites of the MSREs protects the DNA against digestion and therefore can be amplified using PCR. Enzymatic cleavage occurs unhindered at unmethylated restriction sites and subsequent PCR amplification of the target sequence is not observed.


Assuntos
Metilação de DNA , Enzimas de Restrição do DNA/metabolismo , DNA de Plantas/análise , DNA de Plantas/genética , Oryza/genética , Reação em Cadeia da Polimerase/métodos
12.
Methods Mol Biol ; 1991: 101-106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041767

RESUMO

Cell ploidy levels are regulated by developmental and environmental factors and they also impact the outcome of plant microbe interactions. Here we describe a simple and quick procedure to measure cell ploidy levels in Arabidopsis thaliana leaves by flow cytometry. Cell nuclei are isolated by filtering tissue homogenates from chopped plant tissues. DNA in the nuclei is stained by propidium iodide and the fluorescence emitted from the DNA of each nucleus is read by using a flow cytometer. Distribution of ploidy levels within the plant tissues can be calculated based on the distribution of fluorescence signals. Multiple samples can be prepared and analyzed within the same day.


Assuntos
Arabidopsis/genética , Núcleo Celular/genética , DNA de Plantas/genética , Citometria de Fluxo/métodos , Ploidias , Ciclo Celular , DNA de Plantas/análise , Folhas de Planta/genética
13.
Genes (Basel) ; 10(5)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067783

RESUMO

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.


Assuntos
DNA de Plantas/análise , Medicamentos de Ervas Chinesas/análise , Análise de Sequência de DNA/métodos , Proteínas de Cloroplastos/genética , DNA Intergênico/genética , DNA Ribossômico/genética , Combinação de Medicamentos
14.
Anal Bioanal Chem ; 411(14): 3125-3133, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30989272

RESUMO

Huanglongbing (HLB) or citrus greening is a devastating disease of citrus trees that is caused by the gram-negative Candidatus Liberibacter spp. bacteria. The bacteria are phloem limited and transmitted by the Asian citrus psyllid, Diaphorina citri, and the African citrus psyllid, Trioza erytreae, which allows for a wider dissemination of HLB. Infected trees exhibit yellowing of leaves, premature leaf and fruit drop, and ultimately the death of the entire plant. Polymerase chain reaction (PCR) and antibody-based assays (ELISA and/or immunoblot) are commonly used methods for HLB diagnostics. However, they are costly, time-consuming, and destructive to the sample and often not sensitive enough to detect the pathogen very early in the infection stage. Raman spectroscopy (RS) is a noninvasive, nondestructive, analytical technique which provides insight into the chemical structures of a specimen. In this study, by using a handheld Raman system in combination with chemometric analyses, we can readily distinguish between healthy and HLB (early and late stage)-infected citrus trees, as well as plants suffering from nutrient deficits. The detection rate of Raman-based diagnostics of healthy vs HLB infected vs nutrient deficit is ~ 98% for grapefruit and ~ 87% for orange trees, whereas the accuracy of early- vs late-stage HLB infected is 100% for grapefruits and ~94% for oranges. This analysis is portable and sample agnostic, suggesting that it could be utilized for other crops and conducted autonomously. Graphical abstract.


Assuntos
Citrus/química , Citrus/microbiologia , Nutrientes/análise , Doenças das Plantas/microbiologia , Rhizobiaceae/isolamento & purificação , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Citrus/genética , DNA Bacteriano/análise , DNA de Plantas/análise , Ensaio de Imunoadsorção Enzimática , Nutrientes/deficiência , Folhas de Planta/química , Folhas de Planta/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Rhizobiaceae/genética
15.
Methods Mol Biol ; 1963: 31-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875042

RESUMO

Environmental DNA preserved in sediments is rapidly gaining importance as a tool in paleoecology. Sampling procedures for sedimentary ancient DNA (sedaDNA) have to be well planned to ensure clean subsampling of the inside of sediment cores and avoid introducing contamination. Additionally, ancient DNA extraction protocols may need to be optimized for the recovery of DNA from sediments, which may contain inhibitors. Here we describe procedures for subsampling both nonfrozen and frozen sediment cores, and we describe an efficient method for ancient DNA extraction from such samples.


Assuntos
DNA Antigo/análise , DNA Antigo/isolamento & purificação , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Sedimentos Geológicos/análise , Plantas/genética , Manejo de Espécimes/métodos , Ecossistema , Plantas/classificação
16.
Methods Mol Biol ; 1963: 45-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875043

RESUMO

Ancient plant remains from archaeological sites, paleoenvironmental contexts, and herbaria provide excellent opportunities for interrogating plant genetics over Quaternary timescales using ancient DNA (aDNA)-based analyses. A variety of plant tissues, preserved primarily by desiccation and anaerobic waterlogging, have proven to be viable sources of aDNA. Plant tissues are anatomically and chemically diverse and therefore require optimized DNA extraction approaches. Here, we describe a plant DNA isolation protocol that performs well in most contexts. We include recommendations for optimization to retain the very short DNA fragments that are expected to be preserved in degraded tissues.


Assuntos
DNA Antigo/análise , DNA Antigo/isolamento & purificação , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Plantas/genética , Manejo de Espécimes/métodos , Plantas/classificação
17.
PLoS One ; 14(3): e0203737, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30865637

RESUMO

The threat of invasive plant species in island populations prompts the need to better understand their population genetics and dynamics. In the Galapagos islands, this is exemplified by the introduced guava (Psidium guajava), considered one of the greatest threats to the local biodiversity due to its effective spread in the archipelago and its ability to outcompete endemic species. To better understand its history and genetics, we analyzed individuals from three inhabited islands in the Galapagos archipelago with 11 SSR markers. Our results reveal similar genetic diversity between islands, and the populations appear to be distinct: the islands of San Cristobal and Isabela are genetically different while the population of Santa Cruz is a mixture from both. Additional evidence for genetic bottlenecks and the inference of introduction events suggests an original introduction of the species in San Cristobal, from where it was later introduced to Isabela, and finally into Santa Cruz. Alternatively, a second introduction in Isabela might have occurred. These results are contrasted with the historical record, providing a first overview of the history of P. guajava in the Galapagos islands and its current population dynamics.


Assuntos
Variação Genética , Genética Populacional , Genoma de Planta , Espécies Introduzidas , Dinâmica Populacional , Psidium/genética , Biodiversidade , DNA de Plantas/análise , DNA de Plantas/genética , Ecossistema , Equador
18.
Food Chem ; 283: 596-603, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30722917

RESUMO

The spice made from the fruits of Piper nigrum L. (Piperaceae) has high economic value since the beginnings of international trade. Because its price has been increasing, adulterations with papaya seeds, cayenne pepper and maize flour were reported. These have been screened by methodologies dedicated to the detection of single adulterants lacking sensitivity and specificity. Herein we propose a specific, highly-sensitive, high-throughput and affordable qPCR-based methodology for the detection of P. nigrum contaminants (Carica papaya, Zea mays and Capsicum annuum) using plant DNA barcodes trnL and psbA-trnH. The method enables the specific detection of contaminants in a short time with low limits of detection (LOD6 values of 1, 2 and 10 Haploid Genome Equivalents). A market survey (29 samples) revealed 41% of samples contaminated, though about ¾ at very low levels indicating accidental contamination. The proposed tool will contribute to the improvement of quality of this much traded spice.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/análise , Piper nigrum/genética , Capsicum/genética , Carica/genética , Primers do DNA/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , Frutas/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Zea mays/genética
20.
Genes (Basel) ; 10(2)2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736447

RESUMO

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/genética , Análise de Alimentos/métodos , Código de Barras de DNA Taxonômico/normas , DNA de Plantas/análise , Análise de Alimentos/normas , Sequências Repetitivas de Ácido Nucleico , Especiarias/normas , Chá/genética , Chá/normas
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