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DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues
Abstract
DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type.
... Immediately afterwards, 3-5 mL (depending on the size and weight of the insects being investigated) of 2% CTAB (Cetyltrimethylammonium bromide) buffer (2% CTAB, 1% PVP-40, 100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA and 1% Sodium metabisulphite) was added. As for the frass samples, they were prepared differently (Rizzo et al. 2022) allowing for a homogenization phase by using steel jars with a vibromill Mixer Mill MM 400 (Retsch GmbH, Haan, Germany) to which approximately 10 mL of 2% CTAB (Cetyltrimethylammonium bromide) buffer was added. Then, 1 mL of lysate (both from the insects/ larvae and from the frass) was incubated at 65 °C for 5 min. ...
... 1. gDNA Clean Up Kit (Macherey Nagel) as per manufacturer's instructions; 2. FET extraction buffer protocol (Fast extraction TNA) (Edwards et al. 1991); 3. CTAB 2% protocol mentioned above (Rizzo et al. 2022). ...
... The values for the quality and quantity of nucleic acids obtained from the artificial frass were similar to those obtained from the frass of I. sexdentatus. The data revealed the efficiency of the DNA extraction method also in a difficult matrix like frass stored at room temperature, without any particular care (Rizzo et al. 2022). When comparing the three extraction methods, including the one used to discriminate the various matrices under study, the results were all in favour of the CTAB 2% protocol. ...
A molecular tool has been developed for the molecular identification of Ips sexdentatus (Börner 1776) (Coleoptera Curcu-lionidae Scolytidae), the well-known six thooted bark beetle, widely distributed in Eurasia, where it infests several species of the genus Pinus and occasionally a few conifer species of the genera Abies, Larix and Picea. The developed test can be useful both in countries where I. sexdentatus is handled as a quarantine species and, to greater reason, in Europe to discriminate biological traces of this commonly found beetle from those produced by regulated pests. The protocol is based on real-time PCR with TaqMan probe technology and has been developed on whole insect bodies (adults) as well as on artificial frass contaminated by DNA of the beetle. The molecular test developed here for both direct and indirect identification of I. sexdentatus has proven effective in terms of analytical specificity, analytical sensitivity, reliability and reproducibility. The recommended protocol is a practical diagnostic tool allowing a rapid identification of the six toothed bark beetle in the presence of any biological trace of other xylophagous pests collected at points of entry during phytosanitary surveys.
... Sequencing data for the parental lines 'HN53' and 'Zhongbaye' were obtained from a previous study conducted by this research group, referred to as 'sample 19' and 'sample 130' [29], which were sequenced to~10× coverage. Total genomic DNA of 150 F 2 individuals was isolated from leaf tissues using a CTAB procedure as previously described [30], but with modifications. The extracted DNA was submitted for re-sequencing on an Illumina HiSeq 2500 using 150-bp paired-end reads under a 350-bp insert size library, generating 1× coverage data. ...
The species Brassica rapa includes enormous leafy vegetables with extreme leaf morphological diversity. Leaf traits such as size, shape, weight, and ratio of the leaf blade to the petiole contribute to yield, appearance, and desirability to consumers. These leaf-related traits are controlled by quantitative trait loci (QTLs). The construction of high-density bin maps using low-coverage sequencing is a powerful method for QTL fine-mapping and gene identification. In this study, we performed whole-genome re-sequencing of Wutacai ‘Zhongbaye’ and Chinese cabbage ‘HN53’ and 150 F2 individuals to construct a high-density bin map for QTL mapping of 11 leaf-related traits. The parental lines and F2 population were re-sequenced at 10x and 1x coverage, respectively. A map containing 565 bin markers was constructed based on parental single-nucleotide polymorphisms and a modified sliding window approach. The total map length was 944.6 cM and the average distance of the bins was 1.65 cM. In total, 60 significant QTLs controlling 11 leaf-related traits were detected. We further identified candidate genes responsible for these complex leaf-related traits. These findings suggest that this cost-effective bin-mapping approach is capable of rapid identification of QTLs and candidate genes, and will thus facilitate the dissection of the underlying molecular basis of leaf morphological variations and accelerate the improvement of B. rapa vegetable breeding.
Euwallacea similis (Ferrari, 1867), an ambrosia beetle of Asian origin, is recorded for the first time in Europe
on an ‘ornamental’ Ficus macrophylla tree, based on a series of specimens collected in Montechiarugolo (Parma,
Italy), in May 2024. Morphological as well as molecular identifications of this species are presented and debated
and the implications of this finding briefly discussed from a phytosanitary perspective.
For fast and easy isolation of inhibitor-free genomic DNA even from the toughest plant leaf samples, including those high in polyphenols and polysaccharides, a protocol has been developed. To prevent the solubility of polysaccharides in the DNA extract, high salt concentration (1.4 M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) was used for the removal of polyphenols as polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by proteinase K and removed by centrifugation from plant extracts during the isolation process resulting in pure DNA and RNA ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA and RNA isolated from leaves and roots of recalcitrant plants which was free from contamination and color. The average yields of total RNA from roots and shoot of Betula and Grape ranged from 285 to 364 ng/µl with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative reverse transcription polymerase chain reaction, cDNA synthesis and expression analysis. This protocol shown here is reproducible and can be used for a broad spectrum of plant species which have polyphenols and polysaccharide compounds.
Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods--namely three DNA isolation protocols and three commercial DNA isolation kits--were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.
Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.
Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.
A molecular diagnostic method using TaqMan probe qPCR is presented for the identification of Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae) from whole body insects (adults and larvae) and frass samples stored under different conditions. The results showed a perfect amplification of DNA from all samples; the re-peatability and reproducibility of the protocol were very good, with standard deviations of inter-run and intra-run variability less than or equal to 0.5. The assay allowed to discern all A. chinensis samples from those of the other non-target wood-borer species, with 100% correspondence to the homologous sequences. No amplification or cross reactions were observed with A. glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is the most related species among those tested. The protocol was validated by an internal blind panel test which showed a good correspondence between the results obtained by different operators in the same lab. The analytical sensitivity for the lab frass with the Probe qPCR, namely the lowest amount of A. chinensis DNA that can be detected (LoD), was 0.64 pg/µl with a Cq of 34.87. The use of indirect evidence for the identification of a pest is an important feature of the method, which could be crucial to detect the presence of wood-boring insects. This diagnostic tool can help prevent the introduction of A. chinensis into new environments or delimit existing outbreak areas thanks to indirect frass diagnosis.
Wildlife forensic science is a relatively new field in both research and its use in criminal investigations. It should be acknowledged from the outset that wildlife forensic science encompasses many areas of the analytical sciences and each plays a potentially crucial role. This book is focused on the use of DNA in wildlife forensic science while noting the importance of other areas of biological and chemical testing. It is also focused primarily on mammalian and avian DNA typing, again noting the importance and relevance of other species. The book is aimed at those with an interest in DNA for human identification who are considering developing a wildlife forensic science capability. It is also aimed at those with little DNA knowledge but share an interest in wildlife criminal investigations. Only a scant knowledge of DNA is assumed in reading this book and every effort is made to explain terms and concepts.
This chapter leads the reader through the process of designing, testing and validating a species testing. This is conducted using step by step worked examples that can be followed by the user. In this chapter, the phylogenetic definition of a species is used. All members of the same species share a large amount of their DNA. Closely related species share more DNA than more distantly related species; and when two species are more distantly related, then increasing variation is expected to be present between their genomes.
Four DNA extraction protocols were compared,for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known,to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and purification, and the other two were a combination of classical lysis followed by anion exchange,chromatography. The DNA obtained from all procedures was quantified and tested by PCR and Southern hybridisation.Test results indicated superiority of one of the four protocols; a combination,of CTAB lysis followed by anion exchange,chro- matography,which enabled DNA extraction from all seven species. A second protocol also produced DNA from leaves or needles of all species investigated and was well suited for PCR applications but not Southern hybridisations. The remaining protocols produced,DNA from some but not all species tested. Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-
Four DNA extraction protocols were compared for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and purification, and the other two were a combination of classical lysis followed by anion exchange chromatography. The DNA obtained from all procedures was quantified and tested by PCR and Southern hybridisation.Test results indicated superiority of one of the four protocols; a combination of CTAB lysis followed by anion exchange chromatography which enabled DNA extraction from all seven species. A second protocol also produced DNA from leaves or needles of all species investigated and was well suited for PCR applications but not Southern hybridisations. The remaining protocols produced DNA from some but not all species tested.Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-borate-EDTA.
A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.