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The black rot disease in crucifer crops is caused by Xanthomonas campestris pv. campestris (Xcc) which drastically reduces the productivity of crops. Three Xcc races, such as races 1, 4, and 6, have been identified from India that possess nine avr genes, or type-III effectors (T3Es). Here, we used three T3Es—avrXccC, avrBs1, and avrGf1 to identify Xcc from bacterial DNA, bacterial suspensions, Xcc-infected seeds, and the sap of the infected leaves using multiplex PCR. The T3Es were amplified using gene-specific primers with gDNA of Xcc. Then, the multiplex PCR was optimized and amplified T3Es using the sap of black rot-infected cauliflower leaves. Further, this method amplified T3Es from artificially infected seeds (1–100%) of cauliflower and from Xcc colonies (0.1–100%) grown on nutrient agar medium. The primer specificity of T3E genes elucidates that these are specifically detected in all Indian Xcc strains and races, while no bands were observed with other unrelated bacteria, such as X. euvesicatoria, X. oryzae pv. oryzae, Pseudomonas fluorescens, Ralstonia solanacearum, Bacillus subtilis, and B. amyloliquefaciens. Further, this PCR possesses high sensitivity and amplifies T3E genes using up to 0.01 ng Xcc DNA. The high specificity and sensitivity of T3Es-based multiplex PCR make it a potential method and can be used to amplify Xcc from various templates, such as purified DNA, Xcc-infected seeds and leaves, crude extracts, etc., without the need to extract plant or bacterial DNA. [ABSTRACT FROM AUTHOR]

The use of medicinal plants is the basis of traditional healthcare. Recently, the use of herbal medicine has been increasing among consumers due to availability, economy, and less side effect. For instance, the hemiparasite plant Corynaea crassa has medicinal properties and could be found in some regions of America, from Costa Rica to Bolivia. Phytochemical and genetic characterization of medicinal plants is needed for proper identification of metabolites responsible for medicinal properties and for genotyping, respectively. Moreover, characterization of medicinal plants through the use of DNA barcodes is an important tool for phylogenetic analysis and identification of species; furthermore, complemented with phytochemical analysis, both are useful for identification of plant species and quality control of medicinal products. The objective of this study was to analyze the species of C. crassa collected in Ecuador and Peru from the phylogenetic and phytochemical point of view. Polymerase chain reaction (PCR) was performed for amplification of the internal transcribed spacer 1 (ITS1) region after DNA extraction of samples of C. crassa. Blast analysis was performed in the GenBank database with the ITS1 sequences obtained from two accessions of C. crassa from Ecuador (GenBank accession numbers OM471920 and OM471919 for isolates CIBE-17 and CIBE-18, respectively) and three from Peru (GenBank accession numbers OM471921, OM471922, and OM471923 for isolates CIBE-13, CIBE-14, and CIBE-15, respectively). The accessions available in the GenBank were used for phylogenetic analysis. For the phytochemical analysis, hydroalcoholic extracts were obtained by maceration using 80% ethanol as solvent, followed by a derivatization process and analysis by gas chromatography–mass spectrometry. Based on the phylogenetic analysis of the C. crassa samples, the ITS1 sequence could be used to differentiate C. crassa of different locations. The samples of C. crassa from Ecuador and Peru are more similar between them than with other clades including Helosis spp. The phytochemical study revealed differences in the presence and relative abundance of some metabolites; mainly eugenol, 1,4-lactone arabinonic acid, dimethoxyrabelomycin and azelaic acid, which are reported for the first time for the species under study and the genus Corynaea. These results are the first findings on the combined analysis using genetic and phytochemical analysis for C. crassa, which could be used as a useful tool for quality control of the C. crassa species in medicinal products. [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB, also known as "citrus greening"), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus. [ABSTRACT FROM AUTHOR]

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium‐mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer‐DNA (T‐DNA) copies in the host genome. Quantifying the copy number of T‐DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)‐based method to detect a single copy of a T‐DNA insertion in transformants. The competitor line BHK−‐1 that contains a single copy of competitor BHK− (BHK, Basta, Hygromycin, Kanamycin‐resistant genes) was crossed with test transformants and the genomic DNA of F1 plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T‐DNA insertion. We also generated the control lines BHK±1:1 and BHK±2:1, which contain the target (BHK+) and competitor (BHK−) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern‐blot analysis to identify a single copy of T‐DNA insertion in transformants. [ABSTRACT FROM AUTHOR]

The global commercialization of genetically modified (GM) crops has increased the likelihood of feed‐derived transgenic DNA uptake and raised concerns regarding the fate of such DNA fragments in animals consuming a GM diet. The present study applied a novel droplet digital polymerase chain reaction (ddPCR) method to quantitatively detect the concentrations of feed‐derived transgenic (Cry1Ie) and endogenous (rubisco) plant DNA in the livers, intestinal tracts and muscles of zebrafish (Danio rerio) fed GM maize for uantifying feed‐derived DNA in animal tissues as a more sensitive and reliable alternative to qPCR. [ABSTRACT FROM AUTHOR]

Vegan food claims to be free of all animal material. This must be verified by the food control authority and the production control laboratory. The method must be sensitive and able to detect animal material after processing such as pasteurization and storage. We have, therefore, developed a multiplex qPCR to simultaneously detect animal, fish and plant DNA in food samples. The system is cost-efficient and exhibited a high sensitivity. The specificity tests confirmed that all relevant species are detected. The plant-specific system served as a DNA isolation control and was used to generate valid results. A large selection of market samples had been analyzed and showed the expected results and the robustness of this analytical tool in routine analysis. [ABSTRACT FROM AUTHOR]

DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high-throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages – many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases. [ABSTRACT FROM AUTHOR]

The reaction of twelve winter wheat cultivars frequently grown in the Czech Republic and twenty- -five new breeding lines to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in small plot trials from 2017-2018. The assessment was carried out visually by symptoms and by a quantitative real-time polymerase chain reaction (qPCR). The aims of the study were to compare the results of both methods, to evaluate the effect of the resistance gene Pch1 to eyespot, and to select new breeding lines resistant to eyespot. The relationship between the eyespot symptoms and the pathogen DNA content in plant tissues followed a moderate linear regression. Low levels of eyespot were observed in the cultivars/lines possessing the resistance gene Pch1 (Annie, Hermann, Rebell, SG-S1215-14, SG-S1825-14, SG-S791-13) and also in the line SG-SU630-15. The qPCR method was able to detect low levels of the pathogens in the plant tissue and to distinguish two eyespot pathogens. O. acuformis was detected in very low concentrations in the inoculated plants compared with O. yallundae. The eyespot infection rate was significantly higher in 2017 than in the next agricultural season due to extremely dry and warm spring weather in 2018. [ABSTRACT FROM AUTHOR]

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensisspecific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants. [ABSTRACT FROM AUTHOR]

Background: Tea, which is produced from new shoots of existing tea plants (Camellia sinensis), is one of the most popular, non-alcoholic, healthy beverages worldwide. Colletotrichum camelliae is one of the dominant fungal pathogens of tea. The interaction of C. camelliae with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. Currently, many studies characterizing resistance or virulence and aggressiveness use lesion size at the infection sites on the leaves to quantify the growth of the pathogen. However, this method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection. Results: A quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed for the quantification of C. camelliae growth on tea plant. This method was based on the comparison of fungal DNA in relation to plant biomass. This assay was used to investigate the phenotypes of tea plant cultivars in response to C. camelliae infection. Two cultivars, Zhongcha 108 (ZC108) and Longjing 43 (LJ43), were tested with this method. ZC108 was previously reported as an anthracnose-resistant cultivar against C. camelliae, while LJ43 was susceptible. The traditional lesion measurement method showed that both cultivars were susceptible to a virulent strain of C. camelliae, while the qRT-PCR approach indicated that very little fungal growth occurred in the anthracnose-resistant cultivar ZC108. The observed results in this study were consistent with previously published research. In addition, the DNA-based real-time PCR method was applied for analysis of pathogenic differences in general C. camelliae isolates and among several Colletotrichum spp that infect tea. Conclusions: This study showed that the DNA-based qRT-PCR technique is rapid, highly sensitive and easily applicable for routine experiments and could be used in screening for resistant tea plant cultivars or to identify differences in pathogen aggressiveness within and among Colletotrichum species. [ABSTRACT FROM AUTHOR]

Bretziella fagacearum is a devastating disease. In no place in the United States are the losses of oaks in both quantity and diversity worse than in Central Texas. Current management strategies employed in Texas have not proven extremely effective. There is a desperate need to advance every tool possible that can assist those of us who dedicate our lives to the care of oaks. Recently, DNA analysis using polymerase chain reaction (PCR) was proven to be the best standard laboratory protocol in oak-wilt diagnostics (Yang and Juzwik 2017). Specifically, two PCR methods of testing for oak wilt have proven more consistent in finding the oakwilt pathogen in various species within subgenus Quercus section Quercus as well as in section Lobatae (though in this section the results are equivocal). By extension, a reasonable expectation is that this method could more effectively diagnose oak wilt accurately from samples throughout the family Fagaceae. One of the unique and most useful aspects of this method is its ability to detect the fungus in wood samples that are as much as one year old. The fact is this fungus doesn’t stand a chance under the scrutiny of PCR. [ABSTRACT FROM AUTHOR]

Heavy metal pollution, which is one of the most important environmental problems, has a significant effect on plant productivity. Vanadium (V) is considered one of the most important elements of the twenty-first century due to its high consumption in industries. Morphological and physiological measurements have been performed in V studies, but there are deficiencies in molecular studies. The purpose of the present work was to elucidate the effects of V on enzymes activity, DNA damage levels and genomic template stability (GTS%) in Triticum aestivum L., as well as to investigate whether boric acid (BA) has preventive effects on these changes. Antioxidant enzyme activities were determined by SOD (superoxide dismutase), POD (peroxidase) and MDA (malondialdehyde) level. The inter-simple sequence repeats polymerase chain reaction (ISSR-PCR) assay was used to determine the genotoxic effects of V on DNA. According to the obtained results, while V stress increased MDA level and POD enzyme activity, SOD enzyme activity decreased. When V and BA were applied together, MDA level decreased and SOD and POD enzyme activities increased. All doses of V (4.4, 6.6, 8.8 mM) resulted in DNA damage and decreased GTS%. On the other hand, different concentrations of BA (4 and 8 mM) combined with V decreased the toxic effects of V. The results suggested that V could also have negative effects on wheat plants by increasing DNA damage and lipid peroxidation, and BA has an antagonistic effect against V toxicity. BA may be an alternative to reduce genetic damage in plants. [ABSTRACT FROM AUTHOR]

The reaction of twelve winter wheat cultivars frequently grown in the Czech Republic and twenty- -five new breeding lines to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in small plot trials from 2017-2018. The assessment was carried out visually by symptoms and by a quantitative real-time polymerase chain reaction (qPCR). The aims of the study were to compare the results of both methods, to evaluate the effect of the resistance gene Pch1 to eyespot, and to select new breeding lines resistant to eyespot. The relationship between the eyespot symptoms and the pathogen DNA content in plant tissues followed a moderate linear regression. Low levels of eyespot were observed in the cultivars/lines possessing the resistance gene Pch1 (Annie, Hermann, Rebell, SG-S1215-14, SG-S1825-14, SG-S791-13) and also in the line SG-SU630-15. The qPCR method was able to detect low levels of the pathogens in the plant tissue and to distinguish two eyespot pathogens. O. acuformis was detected in very low concentrations in the inoculated plants compared with O. yallundae. The eyespot infection rate was significantly higher in 2017 than in the next agricultural season due to extremely dry and warm spring weather in 2018. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni is worldwide known for its medicinal properties. The objective of this work was to obtain genetic profiles of in vitro grown plants or propagated by cuttings to detect genetic polymorphisms. For this, two DNA extraction protocols were first evaluated, both with modifications. The purified samples were used for random amplification of polymorphic DNA with six pairs of primers. A matrix of data was made from the band patterns. The similarity and genetic distance between both groups of plants were calculated. Genomic DNA was obtained only when the modified protocol proposed by Doyle and Doyle was applied. One of the six primers used was effective for the amplification of DNA segments. In this work, we provide a fast, simple and efficient DNA purification protocol for S. rebaudiana leaf samples. This might be applied for the study of genetic variability and the detection of somaclonal variations using other PCR-based techniques. [ABSTRACT FROM AUTHOR]

Viruses of the genus Begomovirus infect crops of economic importance around the world, including pepper. To date, in Colombia there are no reports of the presence of begomoviruses infecting this crop; therefore, this research work aimed to identify the presence of viruses of this genus in pepper using molecular strategies. Around 197 pepper samples were collected in ten municipalities in Valle del Cauca. Total plant genomic DNA was extracted, and the presence of begomoviruses was detected by using PCR. In order to establish the molecular identity of the virus, fragments of 1.4 kb were amplified from samples collected in Palmira and Vijes municipalities. The fragments obtained were cloned, sequenced, and analyzed. The results show that about 85.7 % of the pepper samples evaluated were positive for begomoviruses. Sequence analysis of the viral fragments of 1.4 kb showed an identity of 91.8 % among them. The nucleotide sequence analysis of the begomoviruses isolated in Vijes and Palmira showed its highest identity values (87.2 % and 86.6 %) with the passion fruit leaf distortion virus, a begomovirus that is affecting passion fruit crops in Colombia. These sequences analyze would indicate that this begomovirus isolated from pepper could be a new species that has not been reported worldwide. To our knowledge, this is the first report of a begomovirus infecting pepper crops in Colombia. [ABSTRACT FROM AUTHOR]

In vitro culture can be used for endangered species preservation but its effect on biochemical properties and genetic stability of plants requires further research. Aim. The study on fatty acids content, phenolic compounds and phylogenetic relationships of Crambe aspera plants upon aseptic cultivation in vitro. Methods. Morphogenic potential of Crambe aspera leaf, root and petiole explants was studied in the Murashige and Skoog (MS) medium with different concentrations of growth factors. Fatty acids (FA) content was determined by gas chromatography- mass spectrophotometry of FA ethers, phenolic compounds were determined by spectrophotometry. Polymerase chain reactions were used to study phylogenetic relationships. Results. The efficient protocols of seed surface sterilization as well as methods of fast microclonal multiplication and obtention of C. aspera callus tissue for were developed. Conclusions. Leaves of both in vitro and in vivo cultured plants had a high content of α-linolenic acid whereas erucic acid was absent. At the same time, the difference in biochemical composition between the plants grown in aseptic and non-aseptic conditions was shown. In vivo populations of C. aspera showed a high level of polymorphism but its genome did not undergo changes after the in vitro establishment. [ABSTRACT FROM AUTHOR]

Amsonia ludoviciana (Louisiana Bluestar), in the Apocynaceae family, is a rare herbaceous perennial plant species found in the ecotones of granite rock outcrops of Georgia as well as the pine flatwoods in Louisiana, Mississippi, and Texas. This research focused on using fluorescently tagged primers to amplify polymorphic microsatellite loci as a way to characterize the population genetic structure of the rare Louisiana Bluestar in Georgia. We sampled 6 populations across the Georgia range of Louisiana Bluestar. We employed 8 microsatellite loci to detect variation within and among Louisiana Bluestar populations and to detect patterns in population structure and dispersal. Values of the genetic-diversity estimator Gst suggested that the majority of the sampled populations demonstrated moderate genetic similarity. Further, Geneland software indicated the presence of 3 clusters. From our population genetic analyses, it can also be inferred that there is substantial gene flow between populations of Louisiana Bluestar in the state of Georgia. [ABSTRACT FROM AUTHOR]

Wood pellets from two port storage silos and four pellet plants (manufacturing facilities) in British Columbia and one pellet plant in Quebec and wood chips used for pellet production from British Columbia were cultured to identify the viable fungi that were present. Living fungal pathogens and decay fungi were cultured from the pre‐production chips, but the fungi isolated from finished pellets were not of phytosanitary concern. The absence of living pinewood nematode in all samples analysed was confirmed by real‐time reverse transcription PCR. Genomic DNA from the pinewood nematode was present in the pre‐pellet chip samples and also in the finished pellets from three of the collection sources. [ABSTRACT FROM AUTHOR]

DNA isolation is a routine procedure when performed in laboratory environment, yet in the field it may still remain problematic. This is especially true of some crop species bred for useful metabolites that may also hinder DNA extraction. Here we compare the efficiency of DNA extraction protocols and commercial DNA isolation kits when used on samples from Helianthus and Allium. Since extraction of DNA is known to be compromised by co-extraction of PCR-inhibiting metabolites, the isolation of DNA was followed by PCR as a testing procedure for the isolation step. The MagnoPrime Fact and MagnoPrime Uni DNA isolation kits were better suited for field work due to faster processing times and smaller required amount of starting material (20 mg fresh/0.5 mg dry). In all cases the subsequent PCR managed to amplify the DNA fragments of interest well enough to be useful in further research. [ABSTRACT FROM AUTHOR]

Metabarcoding of lake sediments have been shown to reveal current and past biodiversity, but little is known about the degree to which taxa growing in the vegetation are represented in environmental DNA (eDNA) records. We analysed composition of lake and catchment vegetation and vascular plant eDNA at 11 lakes in northern Norway. Out of 489 records of taxa growing within 2 m from the lake shore, 17–49% (mean 31%) of the identifiable taxa recorded were detected with eDNA. Of the 217 eDNA records of 47 plant taxa in the 11 lakes, 73% and 12% matched taxa recorded in vegetation surveys within 2 m and up to about 50 m away from the lakeshore, respectively, whereas 16% were not recorded in the vegetation surveys of the same lake. The latter include taxa likely overlooked in the vegetation surveys or growing outside the survey area. The percentages detected were 61, 47, 25, and 15 for dominant, common, scattered, and rare taxa, respectively. Similar numbers for aquatic plants were 88, 88, 33 and 62%, respectively. Detection rate and taxonomic resolution varied among plant families and functional groups with good detection of e.g. Ericaceae, Roseaceae, deciduous trees, ferns, club mosses and aquatics. The representation of terrestrial taxa in eDNA depends on both their distance from the sampling site and their abundance and is sufficient for recording vegetation types. For aquatic vegetation, eDNA may be comparable with, or even superior to, in-lake vegetation surveys and may therefore be used as an tool for biomonitoring. For reconstruction of terrestrial vegetation, technical improvements and more intensive sampling is needed to detect a higher proportion of rare taxa although DNA of some taxa may never reach the lake sediments due to taphonomical constrains. Nevertheless, eDNA performs similar to conventional methods of pollen and macrofossil analyses and may therefore be an important tool for reconstruction of past vegetation. [ABSTRACT FROM AUTHOR]

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is “invisible” by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the “invisible” DNA in botanical extracts. The size of the “invisible” DNA fragments in botanical extracts was around 20–220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future. [ABSTRACT FROM AUTHOR]

The accurate identification and quality evaluation of herbal medical plants is highly necessary to ensure their safety and efficacy. In present study, a new strategy combining DNA barcoding techniques with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) was proposed to facilitate the identification and quality control of M. tenacissima. In present work, the internal transcribed spacer 2 (ITS2) barcode was successfully used to identify 58 M. tenacissima samples and its adulterants. TLC successfully identified the other three M. tenacissima samples that failed to produce ITS2 regions. An adulterant was found in all the 62 samples. Moreover, the content of active medicinal ingredients is important for herbal plants quality. The content of tenacissoside H (TS-H) of M. tenacissima samples was determined by HPLC to range from 0.39% to 1.09%, which meets the criterion of the Chinese Pharmacopoeia. Thus, DNA barcoding coupled with TLC and HPLC is very promising to identify and evaluate the quality of M. tenacissima in the medicine market. [ABSTRACT FROM AUTHOR]

Honey is one of the most frauded food products. Therefore, it is important to develop new analytical systems useful for its authentication. Honey contains intrinsic markers that can be used to identify and monitor its origin, including plant DNA mainly derived by pollen. In this study, we applied a next generation sequencing approach for honey authentication by detecting the prevalent botanical contribution and botanical composition of honeys of different origin. DNA was isolated from nine honeys (six monofloral honeys produced in Italy, two polyfloral honeys produced in East Europe and Chile respectively, and one honeydew honey) and PCR amplified for a chloroplast trnL barcoding fragment. Obtained amplicons were sequenced using the Ion Torrent sequencing platform. Sequence data was interpreted using a customized bioinformatic pipeline that used a reference plant sequence dataset derived by more than 150,000 entries. A total of 254 botanical groups were identified from the nine analysed samples, ranging from 37 groups in orange tree blossom honey to 74 in eucalyptus tree blossom honey. The prevalent expected botanical origin was confirmed in five out of six monofloral honeys. The plant signature of the labelled lime tree blossom honey did not confirm the expected botanical prevalence. The most represented botanical group in the honeydew honey was Castanea . The botanical composition of monofloral and polyfloral honey samples was useful to infer their geographical origin. The metabarcoding based system applied in this study captured the botanical signature of all analysed honey samples and provided information useful for their authentication. [ABSTRACT FROM AUTHOR]

Background: Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications. Results: We developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection. Conclusion: To facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation. [ABSTRACT FROM AUTHOR]

Lycium barbarum, commonly known as goji, is important in Chinese herbal medicine and its fruit is a very important agricultural and biological product. However, the molecular mechanism of formation of its fruit and associated medicinal and nutritional components is unexplored. Moreover, this species lacks SSR markers due to lack of genomic and transcriptomic information. In this study, a total of 139,333 unigenes with average length of 1049 bp and N50 of 1579 bp are obtained by trinity assembly from Illumina sequencing reads. A total of 92,498 (66.38%) unigenes showed similarities in at least one database including Nr (46.15%), Nt (56.56%), KO (15.56%), Swiss-prot (33.34%), Pfam (33.43%), GO (33.62%) and KOG/COG (17.55%). Genes in flavonoid and taurine biosynthesis pathways were found and validated by RT-qPCR. A total of 50,093 EST-SSRs were identified from 38,922 unigenes, and 22,537 EST-SSR primer pairs were designed. Four hundred pairs of SSR markers were randomly selected to validate assembly quality, of which 352 (88%) were successful in PCR amplification of genomic DNA from 11 Lycium accessions and 210 produced polymorphisms. The polymorphic loci showed that the genetic similarity of the 11 Lycium accessions ranged from 0.50 to 0.99 and the accessions could be divided into 4 groups. These results will facilitate investigations of the molecular mechanism of formation of L. barbarum fruit and associated medicinal and nutritional components, and will be of value to novel gene discovery and functional genomic studies. The EST-SSR markers will be useful for genetic diversity evaluation, genetic mapping and marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. [ABSTRACT FROM AUTHOR]

DNA fingerprinting singly or in combination with phytochemical analysis is ideal for quality control of crude plant-based drugs. However, when the source material is tannin rich stem bark, extraction of DNA by conventional methods becomes challenging. In such cases, phytochemical profiling serves as very useful tool for its identification. The work herein described a method for simultaneous DNA isolation and phytochemical extraction for downstream analysis and applications from dried bark powder of Saraca asoca and commercial samples of this crude drug as well as from those of Polyalthia longifolia, its most common adulterant. It is a modified CTAB-based method which involves a pre-extraction step by soaking samples overnight in de-ionized water followed by filtration. The residues in the filter paper were used for DNA isolation and dried filtrate was used for Reverse Phase-High-Performance Liquid Chromatography analysis. Results revealed that genomic DNA isolated was PCR amplifiable with Inter Simple Sequence Repeat and Start Codon Targeted markers. Phenolic compounds of catechin, epicatechin, and gallic acid were detected from the above dried filtrate. The method is simple, reliable and it requires small amount of sample with an option of integrating both phytochemical and DNA-based profiling, from the same starting material. Therefore, the present method could be useful for further potential applications such as quality control assessment of S. asoca products. Graphical abstract: [ABSTRACT FROM AUTHOR]

The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions. [ABSTRACT FROM AUTHOR]

In the recent genomic era, polymerase chain reaction (PCR) has become a basic tool in molecular studies and the success of PCR depends upon the template DNA. PCR technique is quite robust and often unnecessary to extract high quality of DNA and hence crude DNA can be used as template for amplification. Therefore, we have evaluated NaOH-Tris DNA extraction method for PCR analysis because this is very simple, time saving and safe without the need to use expensive or rare materials and laboratory apparatus. This method only requires a small amount of leaf tissue, NaOH, Tris, micro tube and plastic pestle. The amplified PCR products showed clear, sharp and uniform bands gave similar results as compared with the modified CTAB method. The DNA obtained is crude contains impurities like protein, RNA but these impurities did not affect PCR amplification. This DNA extraction method is evaluated for brinjal (Solanum melongena L.), chilli (Capsicum annuum L.), rice (Oryza sativa L.) and tomato (Solanum lycopersicum L.) crop. Many other crop plants could also be amplified using the same DNA extraction method for molecular analysis of large samples. Thus, the use of NaOH-Tris method will allow researchers to obtain DNA from plant quickly for use in molecular studies. [ABSTRACT FROM AUTHOR]

Leaves of melon were collected and extracted by the CTAB method for total DNA which was used for PCR amplification, obtaining the gene sequence of cucumisin promoter. The sequence results were processed and analyzed with DNAman, DNAstar and other softwares, and bioinformatic element analysis was performed with PlatCARE and PLACE. The analysis results showed that the cucumisin promoter shared 100%, 99% and 99% homolog with AY055805, LN713264 and LN681897, respectively. The promoter sequence contains a variety of cis-acting elements common in fruit promoters of higher plants such as TATA-Box and CAAT-Box, and light-responsive elements, some of which involved in ABA and VP1 responsiveness and salicylic acid responsiveness. This study provides a scientific basis for further research on genetic engineering of fruits. [ABSTRACT FROM AUTHOR]

In this study, we report a method to estimate the measurement uncertainty (MU) when testing genetically modified organisms (GMOs) using a bottom-up approach. The repeatability of the test, the linear calibration, the purity of the reference DNA, weighing, the determination of the DNA concentration, and transferring precise volumes with a liquid with pipette were considered MU sources within the real-time quantitative PCR assay of the nopaline synthase terminator. The terminator’s concentration in a mixed sample with an expanded MU was 11.25 ± 1.6%, indicating that 14.2% of the concentration gave an estimated expanded MU. The largest MU originated from pipetting errors and the smallest MU originated from the purity of the reference DNA. Compared with the other sources, the MU from the purity of the reference DNA was negligible. We recommend that the MU be determined when testing food and feed products because of the importance in providing analytical data for GMO threshold compliance decisions. In practice, estimating the MU using the bottom-up approach is suitable for the routine detection of GMOs in a sample in a single testing laboratory. [ABSTRACT FROM AUTHOR]

This paper covers studies on the molecular and ecological aspects of G. glabra var. glandulifera, G. flavescens ssp. flavescens and G. echinata collected from Hatay (Turkey); with the aim to better understand their genetic variation and ecological requirements for possible conservation programs. The material including total genomic DNA was extracted by the CTAB, and for PCR reaction, a total of 14 SSR primers developed for Medicago truncatula were used. PCR amplifications were performed in a Multigen Thermal Cycler. Soil samples were analysed for their texture, pH, total soluble salts, calcium carbonate, total N content, total phosphorus and organic matter content. In order to see the association between genetic, ecological and geographical data, a similarity matrix was generated. Genetic similarity distances between genotypes were correlated with those of Eucledian distances obtained from ecological and geographical data. Analysis of molecular variance (AMOVA) was performed using GenAlEx 6.5 software to determine variation among and within genetic variations. The genetic analysis showed that the highest expected heterozygosity values were obtained from G. glabra while the lowest were obtained from G. echinata. In general heterozygosity values were low, especially for G. echinata. Therefore, variation appears to be lower within each species than among three species. The physical and chemical analysis of soil and plant samples indicates that mineral accumulation in plants is substantially affected by the soil characteristics. There is a need for identification of better strategies for the improvement of varieties, especially for small farmers managing marginal soils. More studies should be conducted in order to safeguard these taxa, especially G. glabra var. glandulifera which is collected intensively due to its economic value, the same is true for endemic taxon G. flavescens ssp. flavescens. [ABSTRACT FROM AUTHOR]

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power. [ABSTRACT FROM AUTHOR]

Background: The isolation of unknown DNA sequences flanked by known sequences is an important task in the event-specific detection of GMOs. None of event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic potato AV43-6-G7. Results: The flanking sequence between the exogenous fragment and recombinant chromosome of this potato was successfully acquired through exogenous gene 5'-RACE. The event-specific primers and Taqman probe were designed to amplify fragments spanning the exogenous DNA and potato genomic DNA. The specific real-time PCR and digital PCR detection methods for AV43-6-G7 potato were established based on primers designed according to the flanking sequences. The detection limit of the qualitative PCR assay was 0.01 % for AV43-6-G7 potato in 100 ng of potato genomic DNA, corresponding to approximately 11.6 copies of the potato haploid genome. The ddPCR assays for Potato AV43-6-G7 achieved a limit of quantification of approximately 58 target copies, with RSD ≤ 25 %. The aLOQ of this system was approximately 1.2 copies. Conclusions: These results indicated that these event-specific methods would be useful for the identification of potato AV43-6-G7. [ABSTRACT FROM AUTHOR]

Background: Phalaenopsis orchid ( Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method. Results: We developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2-5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot. Conclusions: This study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding. [ABSTRACT FROM AUTHOR]

Coffee is one of the main food products commercialized in the world. Its considerable market value among food products makes it susceptible to adulteration, especially with cereals. Therefore, the objective of this study was to develop a method based on Real-Time Polymerase Chain Reaction (PCR) for detection of cereals in commercial ground roast and soluble coffees. After comparison with standard curves obtained by serial dilution of DNA extracted from barley, corn and rice, the method was sensitive and specific to quantify down to 0.6 pg, 14 pg and 16 pg of barley, corn and rice DNA, respectively. To verify the applicability of the method, 30 commercial samples obtained in different countries were evaluated and those classified as gourmets or superior did not present the tested cereals DNA. However, barley was detected in various traditional (cheaper) samples from South America. In addition, corn and rice were also detected in different samples. Real-Time PCR showed to be suitable for detection of food adulterants in commercial ground roast and soluble coffees. [ABSTRACT FROM AUTHOR]

The detection of Genetically Modified (GM) organisms is becoming a legal necessity. This study was carried out to detect genetically modified events in soybeans imported into Iran using simplex and multiplex PCR. Therefore, five samples of imported soybean were obtained from Bandar Imam Khomeini customs. Modified CTAB method was used to extract DNA from soybean seeds. The result indicates that the modified method is suitable for DNA extraction from soybean seeds and probably can be used for other oilseeds. Using specific primers for CaMV 35S promoter, NOS terminator and epsps gene PCR reactions were performed. In this study soybean lectin gene was used as internal control. The results revealed that soybean samples imported from Canada and Paraguay were genetically modified and they had CaMV 35S promoter, NOS terminator and epsps gene in their genomes. The result of simplex PCR was the same as multiplex PCR, but multiplex PCR detected the GM soybeans very quickly and in a cost-saving and timeconsuming way. Based on PCR analysis using GM soybean event-specific primers, it is suggested that the soybean plants may be GTS 40-3-2. No fragment was amplified when the DNA of US or Non-GM soybeans were used as template in the PCR reaction. This is the first report that shows GM soybeans imported to Iran without use of the "GMO" label in the shipment's documentation. [ABSTRACT FROM AUTHOR]

Pine nuts are a known source of food allergens and several cases of adverse immunological reaction after ingestion have been reported. To protect allergic consumers, methods to unequivocally detect the presence of pine nuts in complex matrices must be developed. A Taqman-based real time PCR method for the detection of Pinus spp. was set up. A homemade pesto spiked at known concentration of pine nut powder was used as model food. Moreover, DNA was purified from commercial foods declaring or not the presence of pine nuts. The method displayed a very high efficiency and specificity for the genus Pinus . The intrinsic LOD was 1 pg of DNA, while the practical LOD evaluated on model foods was 0.1 ppm of pine nuts powder, the lowest ever registered for the detection of food allergens via real-time PCR. Finally, the declared presence/absence of pine nut in commercial foods was confirmed. [ABSTRACT FROM AUTHOR]

The traceability of olive oil is an unresolved issue that remains a challenge. In this field, DNA-based techniques are very powerful tools for discrimination that are less negatively influenced by environmental conditions than other techniques. More specifically, quantitative real time PCR (qPCR) achieves a high degree of sensitivity, although the DNA that it can directly isolate from these oils presents drawbacks. Our study reports the analysis of eight systems, in order to determine their suitability for olive detection in oil and oil-derived foodstuffs. The eight systems were analyzed on the basis of their sensitivity and specificity in the qPCR assay, their relative sensitivity to olive DNA detection and DNA mixtures, their sensitivity and specificity to olive in vegetable oils and the detection of olive in commercial products. The results show that the PetN–PsbM system, designed in this study, is a suitable and reliable technique in relation to olive oil and olive ingredients in both food authentication and food safety processes. [ABSTRACT FROM AUTHOR]

Certified reference materials (CRMs) that are compatible with detection methods are needed to detect genetically modified organisms (GMOs). Screening is the first detection step in determining the possible presence of GMO ingredients in food or feed; however, screening has been hindered by the lack of GMO CRMs. In this study, transgenic rice materials were developed via the transformation of a construct harboring 11 commonly used screening elements. Digital PCR was utilized to identify a homozygous single-copy line termed SDrice. The qualitative detections of 11 elements in 21 transgenic materials demonstrated that the genomic DNA of the SDrice was suitable for use as a positive control in the screening of GMO ingredients. The suitability of SDrice as reference material was further checked by testing the sensitivity of 11 known conventional PCR assays, ranging from 10 to 50 copies of the SDrice genome. The standard curves that were created using SDrice DNA series as calibrators all exhibited good linearities in the relationships of the Ct values with the template copy numbers in these 11 real-time PCR assays. The LODs of the real-time PCR assays were estimated to be two to five copies of the SDrice genome. Comparisons of the SDrice with other GM rice revealed that significant differences existed in both the intercepts of the standard curves and the ΔCt values of the exogenous and reference genes for the P-35S, T-nos, HPT, T-35S, and Bar assays; the SDrice was not fit for quantification of other GM rice events. This study provided a matrix reference material (RM) that was suitable for screening GM rice, determination of sensitivity and a LOD of PCR assays, and overcame some of the drawbacks of plasmid DNA as reference material. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg−1 for sesame and 1.4 mg kg−1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products. [ABSTRACT FROM AUTHOR]

Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000-containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 μg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes. [ABSTRACT FROM AUTHOR]

Die-back disease caused by Phomopsis ( Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species-specific PCR-based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species-specific primer-targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313-bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR-based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species-specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages. [ABSTRACT FROM AUTHOR]

To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues (leaves, stems and roots) of Chenopodium quinoa Willd was extracted by modified CTAB method, SDS method and high-salt low-pH method, respectively. The quality and yield of extracted DNA was determined using agarose gel electrophoresis and UV spectrophotometry. At the same time, the PCR-SSR and SSCP molecular detection was also performed. The results showed that the gel test strips, without obvious decomposition, of all the extraction methods were relatively obvious; the genomic DNA yield extracted by modified CTAB method was highest, followed by that by SDS method, and the genomic DNA extracted by high-salt low-pH method was lowest; the genomic DNA yields extracted by different methods from Chenopodium quinoa Willd leaves were all higher than those from roots and stems; the quality of Chenopodium quinoa Willd genomic DNA extracted by modified CTAB method and high-salt low-pH method was better, and polyphenols, polysaccharides and other impurities were removed more completely. The PCR-SSR and SSCP detection results showed that the genomic DNA extracted by different methods from different tissues of Chenopodium quinoa Willd all could be better amplified, and high-quality strips could be obtained. So the Chenopodium quinoa Willd genomic DNA extracted by the three methods all can be used for subsequent molecular biology research. [ABSTRACT FROM AUTHOR]

The oil palm is badly affected by basal stem rot (BSR) disease in Southeast Asia. BSR disease is caused by the fungus Ganoderma boninense, which is a major threat to oil palm compared with other Ganoderma spp. Molecular markers associated with BSR disease will accelerate the identification process of resistant breeding materials in screening of plants for tolerance to the disease at the nursery stage. In this study, 58 simple sequence repeat markers were utilized with three progeny types, namely, KA4G1, KA4G8, and KA14G8, to perform a comparative molecular mapping for association with BSR. A total of 319 alleles were identified with an average of 5.51 alleles per locus. Five markers, mEgCIR0793:180, mEgCIR0894:200, mEgCIR03295:210, mEgCIR3737:146 and mEgCIR3785:299 were found to be associated with Ganoderma disease with P values of 0.018, 0.033, 0.037, 0.034 and 0.037, respectively, in single progeny analysis. However, in pooled data (KA4G1, KA4G8 and KA14G8), only two alleles, mEgCIR0804:213 ( P value = 0.001) and mEgCIR3292:183 ( P value = 0.001), were found to be associated with Ganoderma disease. These analyses confirmed that progeny type KA4G1 was tolerant, whereas the other two were susceptible progeny types. These markers and KA4 progeny will be useful in future works on BSR disease resistance in oil palm. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot of spring oilseed rape ( Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real-time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real-time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear ( R2 > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0-100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real-time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment. [ABSTRACT FROM AUTHOR]

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. [ABSTRACT FROM AUTHOR]

BACKGROUND: There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and livingmodified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events. RESULTS: The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed fromfour endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events. CONCLUSION: The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. [ABSTRACT FROM AUTHOR]

In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products. [ABSTRACT FROM AUTHOR]

Transgenic rice 114-7-2 is a newly developed transgenic rice line of producing human serum albumin (HSA). It has attracted much attention because of its economic potential. This paper was designated to discover the integration site of the transgenic HSA rice line 114-7-2 and to establish event-specific methods for qualitative and quantitative detection of the transgenic HSA rice based on the border junction fragment. One gene fragment of 5′ flanking region was successfully isolated using the TAIL-PCR methods. The fragment sequence showed that a 454-bp junction fragment contained 75 bp of T-DNA sequence and 379 bp of rice genome DNA, which is located in chromosome 4. Event-specific real-time PCR method for HSA rice line 114-7-2 was established with the primers (HSA-F/HSA-R) and the probe (HSA-P) targeting the 454-bp junction region. The qualitative PCR assay showed the limit of detection was 0.01 %. In the event-specific quantitative detection method, the LOQ for 114-7-2 HSA rice was estimated to be 0.025 ng or 50 copies. The method developed in this study is highly specific, sensitive, and reliable for transgenic HSA rice sample detection. [ABSTRACT FROM AUTHOR]