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1.
PLoS One ; 15(3): e0226654, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130218

RESUMO

Although there are many methods for reconstructing diets of the past, detailed taxon identification is still challenging, and most plants hardly remain at a site. In this study, we applied DNA metabarcoding to dental calculus of premodern Japan for the taxonomic identification of food items. DNA was extracted from 13 human dental calculi from the Unko-in site (18th-19th century) of the Edo period, Japan. Polymerase chain reaction (PCR) and sequencing were performed using a primer set specific to the genus Oryza because rice (Oryza sativa) was a staple food and this was the only member of this genus present in Japan at that time. DNA metabarcoding targeting plants, animals (meat and fish), and fungi were also carried out to investigate dietary diversity. We detected amplified products of the genus Oryza from more than half of the samples using PCR and Sanger sequencing. DNA metabarcoding enabled us to identify taxa of plants and fungi, although taxa of animals were not detected, except human. Most of the plant taxonomic groups (family/genus level) are present in Japan and include candidate species consumed as food at that time, as confirmed by historical literature. The other groups featured in the lifestyle of Edo people, such as for medicinal purposes and tobacco. The results indicate that plant DNA analysis from calculus provides information about food diversity and lifestyle habits from the past and can complement other analytical methods such as microparticle analysis and stable isotope analysis.


Assuntos
Arqueologia/métodos , DNA Antigo/isolamento & purificação , Cálculos Dentários/química , Comportamento Alimentar , Oryza/genética , Restos Mortais , Código de Barras de DNA Taxonômico , DNA Fúngico/isolamento & purificação , DNA de Plantas/isolamento & purificação , Feminino , Fungos/genética , História do Século XVIII , História do Século XIX , Humanos , Japão , Masculino , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
2.
PLoS One ; 15(2): e0228776, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032368

RESUMO

The Mediterranean Basin is a biodiversity hotspot, where islands play a key role because of their high biological diversity, degree of endemicity and human pressure. One of these islands, Sardinia, is a good evolutionary laboratory, especially for the study of complex genera, such as Centaurea. In particular, endemic species of Centaurea sect. Centaurea from Sardinia provides an interesting case study of plant evolution on continental islands. We attempted to clarify the processes leading to the diversification of Centaurea species on Sardinia using bi-parentally inherited nuclear markers and maternally inherited plastid markers. Our plastid results revealed the presence of five lineages of sect. Centaurea on the island. Three of them were defined as three species: C. ferulacea, C. filiformis and C. horrida. The other two lineages highlighted the complex evolutionary history of the two polyploids C. corensis and C. magistrorum. Multiple colonization events from the mainland involving the C. deusta and C. paniculata lineages among others, have led to the diversity of sect. Centaurea on Sardinia. One colonization event likely followed a southern path via the land connection between the mainland, the Calabrian Plate and Sardinia. A second pathway likely followed a northern connection, probably through the Tuscan Archipelago. Implications of these findings on conservation efforts for Centaurea endemics on Sardinia are also discussed.


Assuntos
Evolução Biológica , Centaurea/crescimento & desenvolvimento , Centaurea/classificação , Centaurea/genética , DNA de Plantas/química , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Ligação Genética , Ilhas , Itália , Filogenia , Plastídeos/genética , Poliploidia
3.
PLoS Genet ; 16(2): e1008593, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32012153

RESUMO

The repeated evolution of herbicide resistance has been cited as an example of genetic parallelism, wherein separate species or genetic lineages utilize the same genetic solution in response to selection. However, most studies that investigate the genetic basis of herbicide resistance examine the potential for changes in the protein targeted by the herbicide rather than considering genome-wide changes. We used a population genomics screen and targeted exome re-sequencing to uncover the potential genetic basis of glyphosate resistance in the common morning glory, Ipomoea purpurea, and to determine if genetic parallelism underlies the repeated evolution of resistance across replicate resistant populations. We found no evidence for changes in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate's target protein, that were associated with resistance, and instead identified five genomic regions that showed evidence of selection. Within these regions, genes involved in herbicide detoxification-cytochrome P450s, ABC transporters, and glycosyltransferases-are enriched and exhibit signs of selective sweeps. One region under selection shows parallel changes across all assayed resistant populations whereas other regions exhibit signs of divergence. Thus, while it appears that the physiological mechanism of resistance in this species is likely the same among resistant populations, we find patterns of both similar and divergent selection across separate resistant populations at particular loci.


Assuntos
Genoma de Planta/genética , Glicina/análogos & derivados , Herbicidas/farmacologia , Ipomoea/genética , Plantas Daninhas/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/antagonistas & inibidores , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Evolução Molecular , Exoma/genética , Glicina/farmacologia , Resistência a Herbicidas/genética , Ipomoea/efeitos dos fármacos , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Plantas Daninhas/efeitos dos fármacos , Sementes/efeitos dos fármacos , Sementes/genética , Seleção Genética , Análise de Sequência de DNA
4.
Nat Commun ; 11(1): 413, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964885

RESUMO

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent.


Assuntos
Mapeamento Cromossômico/métodos , DNA de Plantas/isolamento & purificação , Haplótipos , Análise de Sequência de DNA/métodos , Vitis/genética , Alelos , DNA de Plantas/genética , Marcadores Genéticos/genética , Genoma de Planta , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites/genética , Filogenia , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único
5.
J Ethnopharmacol ; 247: 112201, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31499140

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.


Assuntos
Benzopiranos/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Variação Genética , Saxifragaceae/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Benzopiranos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , China , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Filogenia , Melhoramento Vegetal , Saxifragaceae/genética , Sementes/genética , Sementes/metabolismo , Temperatura
6.
PLoS One ; 14(12): e0226225, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31826015

RESUMO

Horse-chestnut (Aesculus hippocastanum L.) is an endemic and relict species from the Mediterranean biodiversity hotspot and a popular ornamental tree. Knowledge about the evolutionary history of this species remains scarce. Here, we ask what historical and ecological factors shaped the pattern of genetic diversity and differentiation of this species. We genotyped 717 individuals from nine natural populations using microsatellite markers. The influence of distance, topography and habitat variables on spatial genetic structure was tested within the approaches of isolation-by-distance and isolation-by-ecology. Species niche modeling was used to project the species theoretical range through time and space. The species showed high genetic diversity and moderate differentiation for which topography, progressive range contraction through the species' history and long-term persistence in stable climatic refugia are likely responsible. A strong geographic component was revealed among five genetic clusters that are connected with very limited gene flow. The environmental variables were a significant factor in the spatial genetic structure. Modeling results indicated that future reduction of the species range may affect its survival. The possible impact of climate changes and high need of in situ conservation are discussed.


Assuntos
Aesculus/genética , Variação Genética , Aesculus/fisiologia , Teorema de Bayes , Mudança Climática , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Ecossistema , Fluxo Gênico , Genética Populacional , Genótipo , Grécia , Repetições de Microssatélites , Filogeografia , Refúgio de Vida Selvagem
7.
PLoS One ; 14(12): e0225698, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877137

RESUMO

The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.


Assuntos
Comportamento Alimentar/fisiologia , Avaliação Nutricional , Valor Nutritivo/fisiologia , Ursidae/fisiologia , Animais , DNA de Plantas/isolamento & purificação , Fezes/química , Feminino , Técnicas Genéticas , Masculino , Marmota/genética , Paquistão , Parques Recreativos , Plantas/genética , Fatores Sexuais
8.
Biomol Concepts ; 10(1): 184-193, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31743101

RESUMO

This study was conducted to determine the incidence, diversity and distribution of viruses infecting pepper (Capsicum spp.) in the central, northern and northeastern parts of Thailand. During a survey in 2016 - 2019, a total of 2,149 leaf samples from symptomatic and asymptomatic peppers were collected randomly from farmer's fields, and preliminary tested by an enzyme-linked immunosorbent assay (ELISA) using 7 antibodies specific for cucumber mosaic virus (CMV), chilli veinal mottle virus (ChiVMV), tomato necrotic ringspot virus (TNRV), tobacco mosaic virus (TMV), potato virus Y (PVY), tomato spotted wilt virus (TSWV), and begomoviruses. Our data revealed that the incidence of the viruses infecting pepper in Thailand was high, accounting for nearly 70% (1,482 infected samples). The highest viral incidence was found in the central part (96%), followed by the north (74.4%) and the northeastern (52.8%), respectively. Begomoviruses, CMV, ChiVMV, and TNRV were detected in the samples at varying rates, whereas PVY, TMV, and TSWV were not detected. Of these, the most frequently found virus was Begomoviruses accounting for nearly 33%, with the highest rate (ca. 82%) in the central Provinces of Thailand. In addition, of the 1,482 infected samples, mixed infections among the four viruses were also found in 616 samples (ca. 42%), and CMV + ChiVMV (approximately 11%) was the most common mixed infection. This is the first report describing an occurrence of viruses in pepper of Thailand, and the results obtained have revealed that viruses infecting pepper are widespread, which may pose a threat to pepper production in Thailand.


Assuntos
Begomovirus/isolamento & purificação , Capsicum/virologia , Doenças das Plantas/estatística & dados numéricos , Doenças das Plantas/virologia , Begomovirus/genética , Capsicum/genética , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Tailândia
9.
Nucleic Acids Res ; 47(15): 8050-8060, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31505675

RESUMO

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the µLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/µl for 50 kb fragments and an analytical time of 50 min. Then, µLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with µLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with µLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.


Assuntos
Sistemas CRISPR-Cas , DNA de Plantas/genética , Genoma de Planta/genética , Medicago truncatula/genética , Análise de Sequência de DNA/métodos , Cromossomos Artificiais Bacterianos , Biologia Computacional/métodos , DNA de Plantas/isolamento & purificação , Eletroforese em Gel de Campo Pulsado/métodos , Reprodutibilidade dos Testes
10.
Anal Bioanal Chem ; 411(25): 6583-6590, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422433

RESUMO

This study reports a follow-up investigation on the capture of specific DNA sequences using ion-tagged oligonucleotides (ITOs) and magnetic ionic liquids (MIL). Five allylimidazolium salts bearing octyl substituents ([AOIM+]-ITOs) were used for the selective extraction of the internal transcribed spacer region (ITS) from Arabidopsis thaliana. In this work, the ability of the [AOIM+]-ITOs to enhance the extraction of longer target sequences (~ 700 bp) of plant origin was shown. Moreover, the independence of the probe binding position and the importance of complementarity to the target region for the extraction performance were demonstrated. To test the specificity of the ITOs, the same experiments were performed using the ITS region from another plant species, with a lower target capture for the probes which were specific for the A. thaliana sequence. Finally, extraction in the presence of interferences (heterogenous DNA, primary and secondary metabolites, proteins) provided interesting and insightful results. This work illustrates the feasibility and versatility of these probes when coupled to MILs for rapid, cost-effective, and environmentally sensitive sample preparation in the extraction of specific target sequences from different origins. Graphical abstract.


Assuntos
Arabidopsis/química , DNA Intergênico/isolamento & purificação , DNA de Plantas/isolamento & purificação , Líquidos Iônicos/química , Imãs/química , Arabidopsis/genética , Sequência de Bases , DNA Intergênico/genética , DNA de Plantas/genética , Imidazóis/química , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética
11.
C R Biol ; 342(5-6): 142-153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447175

RESUMO

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC2F2 was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.


Assuntos
Cruzamentos Genéticos , Melhoramento Genético/métodos , Oryza/genética , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Seleção Genética/genética , Aspergillus oryzae , Cruzamento , Culinária , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Resistência à Doença , Marcadores Genéticos , Genoma de Planta/genética , Índia
13.
Food Chem ; 300: 125205, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31330372

RESUMO

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.


Assuntos
DNA de Plantas/isolamento & purificação , Nanopartículas/química , Óleos Vegetais/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Verduras/genética , Fracionamento Químico/métodos , Cloroplastos/genética , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Polimetil Metacrilato/química , Ribulosefosfatos/genética , Dióxido de Silício
14.
C R Biol ; 342(5-6): 154-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31239197

RESUMO

Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease of wheat worldwide, including India. Growing resistant cultivars is the most cost-effective and eco-friendly approach to manage the disease. In this study, 70 publically available molecular markers were used to identify the distribution of 35 Yr genes in 68 wheat genotypes. Out of 35 Yr genes, 25 genes amplified the loci associated with Yr genes. Of the 35, 18 were all-stage resistance ASR (All-stage resistance) genes and 7 (Yr16, Yr18, Yr29, Yr30, Yr36, Yr46 &Yr59) were APR (Adult-plant resistance) genes. In the field tests, evaluation for stripe rust was carried out under artificial inoculation of Pst. Fifty-three wheat genotypes were found resistant to yellow rust (ITs 0), accounting for 77.94% of total entries. Coefficients of infection ranged from 0 to 60 among all wheat genotypes. Two genotypes (VL 1099 & VL 3002) were identified with maximum 15 Yr genes followed by 14 genes in VL 3010 and HI8759, respectively. Maximum number of all-stage resistance genes were identified in RKD 292 (11) followed by ten genes in DBW 216, WH 1184 and VL 3002. Maximum number of adult-plant resistance gene was identified in VL 3009 (6), HI 8759 (5) and Lassik (4) respectively. Genes Yr26 (69.2%), Yr2 (69.1%), Yr64 (61.7%), Yr24 (58.9%), Yr7 (52.9%), Yr10 (50%) and Yr 48 (48.5%) showed high frequency among selected wheat genotypes, while Yr9 (2.94%), Yr36 (2.94%), Yr60 (1.47%) and Yr32 (8.8%) were least frequent in wheat genotypes. In future breeding programs, race specific genes and non-race specific genes should be utilised to pyramid with other effective genes to develop improved wheat cultivars with high-level and durable resistance to stripe rust. Proper deployment of Yr genes and utilizing the positive interactions will be helpful for resistance breeding in wheat.


Assuntos
Resistência à Doença/genética , Doenças das Plantas/prevenção & controle , Triticum/genética , Basidiomycota , Cruzamento , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Genes de Plantas , Marcadores Genéticos , Genótipo , Índia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia
15.
Mol Biol Rep ; 46(4): 4611-4615, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31073915

RESUMO

Despite the importance in conservation and breeding purposes, molecular studies using Cerrado plant species are still rare, mainly because of their high amounts of secondary compounds, impeding DNA extraction to downstream applications, such as PCR amplification. To date, the DNA extraction methods described are sometimes inadequate for these species, expensive, time-intensive and/or use very toxic reagents. Here, we present a simple and effective method, based on SDS and Triton X-100, to obtain high DNA quality and quantity from Cerrado plant species for molecular biological techniques. The DNA obtained by our protocol was free of contaminants and excellent for enzymatic restriction and PCR amplification. The concentration of extracted DNA across all tested species ranged from 156 to 1166 ng µL-1 (1.56-11.66 µg g-1 of dry tissue), with an A260/A280 ratio from 1.78 to 1.92. The new DNA extraction protocol described here provides high DNA quality and quantity from Cerrado plant species in a fast, simple and less toxic way. Thus, the use of our method will allow ecologists, geneticists and breeders to rapidly obtain high-quality and -quantity DNA from Cerrado plant species for any molecular biology study.


Assuntos
DNA de Plantas/isolamento & purificação , DNA/isolamento & purificação , Brasil , Plantas/genética , Reação em Cadeia da Polimerase/métodos
16.
Viruses ; 11(4)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987196

RESUMO

Plant-viroid interactions represent a valuable model for delineating structure-function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.


Assuntos
Arabidopsis/citologia , Biologia Molecular/métodos , Protoplastos/virologia , Viroides/fisiologia , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Genoma de Planta , Interações Hospedeiro-Patógeno , Protoplastos/metabolismo , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Viroides/genética , Replicação Viral
17.
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
18.
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
19.
PLoS One ; 14(2): e0211471, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707722

RESUMO

Miscanthus lutarioriparius is a native perennial Miscanthus species of China, which is currently used as raw material of papermaking and bioenergy crop. It also has been considered as a promising eco-bioindustrial plant, which can offer raw material and gene for the biomass industry. However, lack of germplasm resources and genetic diversity information of M. lutarioriparius have become the bottleneck that prevents the stable and further development of the biomass industry. In the present study, genetic diversity of 153 M. lutarioriparius individuals nine populations was studied using 27 Start Codon Targeted (SCoT) markers. High polymorphic bands (97.67%), polymorphic information content (0.26) and allele number (1.88) showed SCoT as a reliable marker system for genetic analysis in M. lutarioriparius. At the species, the percentage of polymorphic loci [PPL] was 97.2%, Nei's gene diversity [H] was 0.36, Shannon index [I] was 0.54 and Expected Heterozygosity [He] was 0.56. Genetic variation within populations (84.91%) was higher than among populations (15.09%) based on analysis of molecular variance (AMOVA). Moderate level of genetic differentiation was found in M. lutarioriparius populations (Fst = 0.15), which is further confirmed by STRUCTURE, principal coordinates analysis (PCoA) and an unweighted pair group method with arithmetic mean (UPGMA) analysis that could reveal a clear separation between groups of the north and south of Yangtze River. The gene flow of the populations within the respective south and north of Yangtze River area was higher, but lower between the areas. There was no obvious correlation between genetic distance and geographic distance. The breeding systems, geographical isolation and fragmented habitat of M. lutarioriparius may be due to the high level of genetic diversity, moderate genetic differentiation, and the population, structure. The study further suggests some measure for conservation of genetic resources and provides the genetic basis for improving the efficiency of breeding based on the results of diversity analysis.


Assuntos
Variação Genética , Poaceae/genética , Teorema de Bayes , China , Análise por Conglomerados , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Fluxo Gênico , Repetições de Microssatélites/genética , Polimorfismo Genético , Análise de Componente Principal
20.
Anal Bioanal Chem ; 411(11): 2461-2469, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30810790

RESUMO

Accurate quantitative methods are needed to determine the amount of transgenic material in ingredients and comply with labelling GMO thresholds. Quantitative real-time PCR methods are usually applied for GMO quantification, but since a few years, digital PCR (dPCR) has been described as a potential alternative by quantifying DNA molecules directly without any standard curves. In this study, the performance of dPCR to quantify 9 GM-soya events and 15 GM-maize events was assessed. Following GMO validation guidelines, the trueness and precision were determined on high, medium and low levels of transgenic content. Results showed biases below ± 25% and satisfactory precision data. Limits of quantification were determined for each GM-event and were between 12 and 31 target copies. The reliability of GMO quantification by dPCR was further confirmed by analysing several proficiency test samples. Overall, dPCR showed accurate and precise GMO quantification on all the tested GM-events, from high to low transgenic amount. With its ease-of-use, dPCR was found to be an appealing alternative technology for routine GMO testing laboratories. Graphical abstract.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/métodos , Soja/genética , Zea mays/genética , DNA de Plantas/isolamento & purificação , Limite de Detecção , Reprodutibilidade dos Testes
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