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1.
J Agric Food Chem ; 68(3): 899-906, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31891505

RESUMO

Convenient, portable, and low-cost multiplex nucleic acid testing (NAT) systems are the trends in the fields of food safety, environmental microorganisms, molecular diagnosis, etc. In this study, we developed a novel system for visual monitoring of multiple nucleic acids combining a mini-disk capillary array (diameter = 17 mm, embedded with 6-10 capillaries), visual loop-mediated isothermal amplification (LAMP), and quick DNA extraction called mDC-LAMP. The performance and applicability of mDC-LAMP in testing multiple nucleic acids were evaluated and verified employing genetically modified contents analysis as an example. All of the results confirmed that mDC-LAMP has the advantages of high specificity without any cross contamination, high sensitivity with a limit of detection of 25 copies/reaction, high throughput with flexible channel sensors, easy fabrication, and low costs. We believe that mDC-LAMP is a competitive choice for on-spot monitoring of multiple nucleic acids in terms of the easy fabrication/operation, low costs, and suitable performance presented in the nucleic acids test.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , DNA de Plantas/genética , Alimentos Geneticamente Modificados , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase , Zea mays/genética
2.
Gene ; 726: 144154, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31589962

RESUMO

In this work the complete chloroplast DNAs of Allium paradoxum and Allium ursinum, two edible species of Allium subg. Amerallium (the first lineage), were sequenced, assembled, annotated, and compared with complete Allium plastomes of the second and third evolutionary lines from GenBank database. The A. ursinum plastome contains 90 predicted genes (81 unique) including 5 pseudogenes, while A. paradoxum has 88 predicted genes (79 unique) including 19 pseudogenes. The comparative analysis has revealed that the A. paradoxum plastome differs markedly from those of other species. Due to many deletions, the A. paradoxum plastome is the shortest of known for Allium species, being only 145,819 bp long. The most prominent distinctions are (1) a 4825 bp long local inversion that spans from the ndhE to the rpl32 gene in the small single copy region and (2) pseudogenization, or the loss of all NADH-genes. In contrast, the plastome of A. ursinum - a species from the first evolutionary line (as well as A. paradoxum) - resembles the Allium species of the second and third evolutionary lines, showing no large rearrangements or discrepancies in gene content. It is unclear yet whether only A. paradoxum was affected by some evolutionary events or its close relatives from both sect. Briseis and other sections of Amerallium were altered as well. We speculate the sunlight-intolerant, shade-loving nature of A. paradoxum and the impairment of the ndh genes in its plastome could be interrelated phenomena.


Assuntos
Allium/genética , Rearranjo Gênico/genética , Genes de Plantas/genética , Cebolas/genética , DNA de Cloroplastos/genética , DNA de Plantas/genética , Evolução Molecular , Genoma de Cloroplastos/genética , Genoma de Planta/genética , Filogenia , Folhas de Planta/genética , Pseudogenes/genética , Análise de Sequência de DNA/métodos
3.
J Forensic Sci ; 65(1): 52-60, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31433500

RESUMO

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used "legal high" opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/µL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen® PLUS.


Assuntos
Cannabis/genética , DNA de Plantas/genética , Toxicologia Forense/métodos , Mitragyna/genética , Primers do DNA , Eletroforese em Gel de Ágar , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Temperatura de Transição
4.
Biosci Biotechnol Biochem ; 84(1): 43-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31495297

RESUMO

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/genética , Sequência de Bases/genética , Percepção de Cores , Primers do DNA/genética , Enzimas de Restrição do DNA/genética , Eletroforese em Gel de Ágar , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Amplificação de Genes , Limite de Detecção , Compostos Orgânicos/química , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Temperatura Ambiente , Tempo
5.
Talanta ; 206: 120220, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514891

RESUMO

This work addresses a technological advance applied to the construction of a magnetogenoassay with electrochemical transduction for the maize taxon-specific (HMGA gene) detection using gold-coated magnetic nanoparticles as nanosized platform. Superparamagnetic core-shell Fe3O4@Au nanoparticles (10.4 ±â€¯1.7 nm) were used to assemble the genoassay through the covalent immobilization of HMGA DNA probes onto carboxylated self-assembled monolayers at the nanoparticles surface. A hybridization reaction using sandwich format was selected to prevent inefficient hybridization connected with stable secondary DNA structures using also fluorescein isothiocyanate as DNA signaling tag. The labelling of the hybridization reaction with enzymes allowed the chronoamperometric measurement of the peroxidase activity linked to the nanoplatform located on gold surface. Using this electrochemical magnetogenoassay a linear concentration range from 0.5 to 5 nM and a LOD of 90 pM with a RSD <1.2% was calculated. Certified maize was evaluated without further purification after PCR amplification. This work highlights the efficacy of the electrochemical magnetogenoassay for the HMGA detection, showing its potential as alternative procedure for the verification of the compliance of the legislation.


Assuntos
Técnicas Biossensoriais/métodos , Genes de Plantas , Ouro/química , Proteínas HMGA/genética , Nanopartículas de Magnetita/química , Zea mays/genética , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Técnicas Eletroquímicas/métodos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
6.
Nat Commun ; 10(1): 4680, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615981

RESUMO

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.


Assuntos
Frutas/química , Phoeniceae/genética , Pigmentação/genética , Processos de Determinação Sexual/genética , Alelos , Mapeamento Cromossômico , Códon de Iniciação , DNA de Plantas/genética , Frutose , Frutas/genética , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Glucose , Mutação , Fenótipo , Polimorfismo Genético , Retroelementos , Análise de Sequência de DNA , Amido , Sacarose , beta-Frutofuranosidase/genética
7.
Zhongguo Zhong Yao Za Zhi ; 44(17): 3615-3621, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31602931

RESUMO

The type and frequency of simple sequence repeats( SSRs) in the genomes was investigated using the DNA sequence data of Pueraria lobata and P. thomsonii. Based on these SSRs,20 pairs of SSR primers were designed and 5 high polymorphism primer pairs were selected to analyze genetic diversity of 9 cultivars of P. thomsonii in Jiangxi province. The results showed that the 5 pairs of primers could generate 16 polymorphic alleles bands. The average polymorphism information content( PIC) of each SSR primer pair was 0. 600 7.According to the genetic similarity coefficients,the 9 cultivars of P. thomsonii can be classified into 6 germplasms. This study established DNA identity cards with 5 pairs of SSR primers for different germplasm resources of P. thomsonii in Jiangxi province,which provided reference information for the selection of fine germplasms of P. thomsonii and the theoretical basis for the study of Dao-di herbs.


Assuntos
DNA de Plantas/genética , Repetições de Microssatélites , Pueraria/genética , China , Genômica , Polimorfismo Genético
8.
Theor Appl Genet ; 132(12): 3347-3355, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31583438

RESUMO

KEY MESSAGE: Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.


Assuntos
Azacitidina/farmacologia , Elementos de DNA Transponíveis , Oryza/genética , Mapeamento Cromossômico , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Oryza/efeitos dos fármacos , Fenótipo , Sementes/genética
9.
Cytogenet Genome Res ; 159(1): 48-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31610539

RESUMO

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.


Assuntos
Amaryllidaceae/genética , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Replicação do DNA/genética , Zea mays/genética , Cromatina/metabolismo , Cromossomos/genética , DNA de Plantas/genética , Endonucleases/genética , Hibridização in Situ Fluorescente/métodos , RNA Guia/genética , Telômero/genética
10.
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
11.
Molecules ; 24(18)2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500338

RESUMO

Fritillariae Bulbus is a precious Chinese herbal medicine that is grown at high elevation and used to relieve coughs, remove phlegm, and nourish the lungs. Historically, Fritillariae Bulbus has been divided into two odourless crude drugs: Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus. However, now the Chinese Pharmacopoeia has described five Fritillariae Bulbus-the new additions include Fritillariae Pallidiflorae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Hupehensis Bulbus. Because the morphology of dried Fritillariae Bulbus is similar, it is difficult to accurately identify the different types of Fritillariae Bulbus. In the current study, we develop a method combining DNA barcoding and high-performance liquid chromatography (HPLC) to help distinguish Fritillariae Cirrhosae Bulbus from other Fritillariae Bulbus and guarantee species traceability of the five types of Fritillariae Bulbus. We report on the validation of an integrated analysis method for plant species identification using DNA barcoding that is based on genetic distance, identification efficiency, inter- and intra-specific variation, calculated nearest distance, neighbour-joining tree and barcoding gap. Our results show that the DNA barcoding data successfully identified the five Fritillariae Bulbus by internal transcribed spacer region (ITS) and ITS2, with the ability to distinguish the species origin of these Fritillariae Bulbus. ITS2 can serve as a potentially useful DNA barcode for the Fritillaria species. Additionally, the effective chemical constituents are identified by HPLC combined with a chemical identification method to classify Fritillaria. The HPLC fingerprint data and HCA (hierarchical clustering analysis) show that Fritillariae Cirrhosae Bulbus is clearly different from Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus, but there is no difference between Fritillariae Cirrhosae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Pallidiflorae Bulbus. These results show that DNA barcoding and HPLC fingerprinting can discriminate between the five Fritillariae Bulbus types and trace species to identify related species that are genetically similar.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Medicamentos de Ervas Chinesas/química , Fritillaria/classificação , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , DNA Mitocondrial/genética , DNA de Plantas/genética , Fritillaria/química , Fritillaria/genética , Filogenia , Análise de Sequência de DNA
12.
Plant Sci ; 287: 110189, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481211

RESUMO

Microspores exposed to some stress factors may display cell totipotency and could be reprogrammed towards embryogenic development. Plant breeding and genetic engineering widely use haploids/doubled haploids (DHs) derived from in vitro-cultured microspores, but the mechanism of this process remains poorly understood. Recently published data suggest that microspore embryogenesis (ME) is accompanied by changes in DNA methylation and chromatin reorganization. Here, we used two triticale DH lines (DH19 and DH28), significantly different with respect to embryogenic potential. To change DNA methylation levels, we applied two cytosine-analogs: 5-azacytidine (AC) and 2'-deoxy-5-azacytidine (DAC) treatments. We found that chemically-induced DNA demethylation caused chromatin relaxation and dysregulation of marker genes (TaTPD1-like, GSTF2, GSTA2, CHI3, Tad1, TaNF-YA7, SERK2, TaME1) related to ME. Both drugs showed significant cytotoxicity in a dose-dependent manner. We noticed that lines varied in terms of overall DNA methylation levels and responded in a different way to hypomethylation caused by the drugs. DH19 (low embryogenic) after inhibitors treatment, showed higher microspore viability, but its recalcitrancy was not overcome. For highly embryogenic DH28, we noted significantly higher effectiveness of embryo-like structure production and plant regeneration. In summary, our study provides new insight into the role of DNA methylation in ME initiation. They suggest potential benefits resulting from the utilization of epigenetic inhibitors to improve the process of DHs production.


Assuntos
Triticale/genética , Cruzamento , Metilação de DNA , DNA de Plantas/genética , Haploidia , Pólen/embriologia , Pólen/genética , Triticale/embriologia
13.
Plant Sci ; 287: 110174, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481216

RESUMO

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.


Assuntos
Chrysanthemum/genética , Chrysanthemum/ultraestrutura , DNA de Plantas/genética , Diploide , Flores/ultraestrutura , Hibridização Genética , Microscopia Eletrônica de Varredura , Modelos Biológicos , Mutação , Filogenia , Melhoramento Vegetal/métodos , Polinização , Autofertilização
14.
Nat Commun ; 10(1): 3916, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477705

RESUMO

Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA de Plantas/química , RNA de Plantas/genética
15.
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
16.
Mol Cell ; 75(3): 576-589.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398324

RESUMO

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.


Assuntos
Proteínas de Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Replicase/genética , Ribonuclease III/genética , Transcrição Genética , Arabidopsis/genética , Proteínas Argonauta/genética , Metilação de DNA/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
17.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370193

RESUMO

Prior experiments illustrated reactive oxygen species (ROS) overproduction in maize plants infested with bird-cherry-oat (Rhopalosiphum padi L.) aphids. However, there is no available data unveiling the impact of aphids feeding on oxidative damages of crucial macromolecules in maize tissues. Therefore, the purpose of the current study was to evaluate the scale of oxidative damages of genomic DNA, total RNA and mRNA, proteins, and lipids in seedling leaves of two maize genotypes (Zlota Karlowa and Waza cvs-susceptible and relatively resistant to the aphids, respectively). The content of oxidized guanosine residues (8-hydroxy-2'-deoxyguanosine; 8-OHdG) in genomic DNA, 8-hydroxyguanosine (8-OHG) in RNA molecules, protein carbonyl groups, total thiols (T-SH), protein-bound thiols (PB-SH), non-protein thiols (NP-SH), malondialdehyde (MDA) and electrolyte leakage (EL) levels in maze plants were determined. In addition, the electrical penetration graphs (EPG) technique was used to monitor and the aphid stylet positioning and feeding modes in the hosts. Maize seedlings were infested with 0 (control), 30 or 60 R. padi adult apterae per plant. Substantial increases in the levels of RNA, protein and lipid oxidation markers in response to aphid herbivory, but no significant oxidative damages of genomic DNA, were found. Alterations in the studied parameters were dependent on maize genotype, insect abundance and infestation time.


Assuntos
Afídeos/fisiologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Parasita/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Zea mays/genética , /metabolismo , Animais , Afídeos/patogenicidade , DNA de Plantas/genética , DNA de Plantas/metabolismo , Genótipo , Guanosina/análogos & derivados , Guanosina/metabolismo , Lipídeos/química , Malondialdeído/metabolismo , Oxirredução , Estresse Oxidativo , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/genética , Plântula/parasitologia , Compostos de Sulfidrila/metabolismo , Zea mays/parasitologia
18.
BMC Biotechnol ; 19(1): 55, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370820

RESUMO

BACKGROUND: Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. RESULTS: We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal. CONCLUSIONS: Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.


Assuntos
Produtos Agrícolas/genética , Primers do DNA/genética , DNA de Plantas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Regiões Promotoras Genéticas/genética , Sequência de Bases , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Homologia de Sequência do Ácido Nucleico
19.
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
20.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426439

RESUMO

Epimedium wushanense (Berberidaceae) is recorded as the source plant of Epimedii Wushanensis Folium in the Chinese Pharmacopoeia. However, controversies exist on the classification of E. wushanense and its closely related species, namely, E. pseudowushanense, E. chlorandrum, E. mikinorii, E. ilicifolium, and E. borealiguizhouense. These species are often confused with one another because of their highly similar morphological characteristics. This confusion leads to misuse in the medicinal market threatening efficiency and safety. Here, we studied the plastid genomes of these Epimedium species. Results show that the plastid genomes of E. wushanense and its relative species are typical circular tetramerous structure, with lengths of 156,855-158,251 bp. A total of 112 genes were identified from the Epimedium plastid genomes, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A loss of rpl32 gene in E. chlorandrum was found for the first time in this study. The phylogenetic trees constructed indicated that E. wushanense can be distinguished from its closely related species. E. wushanense shows a closer relationship to species in ser. Dolichocerae. In conclusion, the use of plastid genomes contributes useful genetic information for identifying medicinally important species E. wushanense and provides new evidence for understanding phylogenetic relationships within the Epimedium genus.


Assuntos
Epimedium/genética , Genomas de Plastídeos , DNA de Plantas/genética , Epimedium/classificação , Genômica , Filogenia , Plastídeos/classificação , Plastídeos/genética
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